Compositions and methods for inhibiting the expression of anti-apoptopic genes

ABSTRACT

The present invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of an anti-apoptotic gene, comprising an antisense strand having a nucleotide sequence which is less that 25 nucleotides in length and which is substantially complementary to at least a part of an apoptotic gene, such as a Bcl gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by the expression of an anti-apoptotic gene using the pharmaceutical composition; and methods for inhibiting the expression of an anti-apoptotic gene in a cell.

RELATED APPLICATIONS

This application is a divisional application which claims priority toU.S. patent application Ser. No. 11/229,183, filed Sep. 15, 2005, whichis a continuation in part of U.S. application Ser. No. 10/941,663, filedSep. 15, 2004, which is a continuation-in-part of U.S. application Ser.No. 10/384,260, filed Mar. 7, 2003, which is a continuation-in-part ofInternational Application No. PCT/EP02/00151, which designated theUnited States and was filed Jan. 9, 2002, and which claims the benefitof German Patent No. 101 00 586.5, filed Jan. 9, 2001. The entireteachings of the above applications are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use in mediating RNA interference to inhibit the expression of ananti-apoptotic target gene, such as a Bcl gene.

BACKGROUND OF THE INVENTION

Many diseases, including cancers, arise from the abnormalover-expression or -activity of a particular gene, a group of genes, ora mutant form of protein. The therapeutic benefits of being able toselectively silence the expression of these genes is obvious.

A number of therapeutic agents designed to inhibit expression of atarget gene have been developed, including antisense ribonucleic acid(RNA) (see, e.g., Skorski, T. et al., Proc. Natl. Acad. Sci. USA (1994)91:4504-4508) and hammerhead-based ribozymes (see, e.g., James, H. A,and I. Gibson, Blood (1998) 91:371). However, both of these agents haveinherent limitations. Antisense approaches, using either single-strandedRNA or DNA, act in a 1:1 stoichiometric relationship and thus have lowefficacy (Skorski et al., supra). Hammerhead ribozymes, which because oftheir catalytic activity can degrade a higher number of targetmolecules, have been used to overcome the stoichiometry problemassociated with antisense RNA. However, hammerhead ribozymes requirespecific nucleotide sequences in the target gene, which are not alwayspresent.

More recently, double-stranded RNA molecules (dsRNA) have been shown toblock gene expression in a highly conserved regulatory mechanism knownas RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the useof a dsRNA of at least 25 nucleotides in length to inhibit theexpression of a target gene in C. elegans. dsRNA has also been shown todegrade target RNA in other organisms, including plants (see, e.g., WO99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.),Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000)10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00586.5, Kreutzer et al.).

Briefly, the RNA III Dicer enzyme processes dsRNA exceeding a certainlength into small interfering RNA (siRNA) of approximately 22nucleotides. One strand of the siRNA (the “guide strand”) then serves asa guide sequence to induce cleavage of messenger RNAs (mRNAs) comprisinga nucleotide sequence which is at least partially complementary to thesequence of the guide strand by an RNA-induced silencing complex RISC(Hammond, S. M., et al., Nature (2000) 404:293-296). The guide strand isnot cleaved or otherwise degraded in this process, and the RISCcomprising the guide strand can subsequently effect the degradation offurther mRNAs by sequence specific cleavage. In other words, RNAiinvolves a catalytic-type reaction whereby new siRNAs are generatedthrough successive cleavage of long dsRNA. Thus, unlike antisense, RNAidegrades target RNA in a non-stoichiometric manner. When administered toa cell or organism, exogenous dsRNA has been shown to direct thesequence-specific degradation of endogenous messenger RNA (mRNA) throughRNAi.

Gautschi et al. report that the expression levels of the anti-apoptoticproteins Bcl-1 and Bcl-xL are elevated during the development andprogression of tumors (Gautschi, O., et al., J. Natl. Cancer Inst.(2001) 93:463-471). Tumor growth (but not size) was reduced byapproximately 50-60% in nude mice treated with a combination ofsingle-stranded antisense oligoribonucleotides targeted to Bcl-2 andBcl-xL genes. However, because of the 1:1 stoichiometric relationshipand thus low efficiency of antisense RNA, the anti-Bcl treatmentrequired 20 milligrams of antisense RNA per kilogram body weight ofrecipient mouse per day. Producing therapeutically sufficient amounts ofRNA is not only expensive, but single-stranded antisense RNA is highlysusceptible to degradation by serum proteases, thus resulting in a shortin vivo half-life.

Despite significant advances in the field, there remains a need for anagent that can selectively and efficiently silence a target gene usingthe cell's own RNAi machinery. More specifically, an agent that has bothhigh biological activity and in vivo stability, and that can effectivelyinhibit expression of a target anti-apoptotic gene at a low dose, wouldbe highly desirable. Compositions comprising such agents would be usefulfor treating diseases caused by the expression of these genes.

SUMMARY OF THE INVENTION

The present invention discloses double-stranded ribonucleic acid(dsRNA), as well as compositions and methods for inhibiting theexpression of a target gene, such as an anti-apoptotic gene, in a cellusing the dsRNA. The present invention also discloses compositions andmethods for treating diseases caused by the expression of a targetanti-apoptotic gene (e.g., a Bcl gene). The dsRNA of the inventioncomprises an RNA strand (the antisense strand) having a region which isless than 30 nucleotides in length and is substantially complementary toat least part of an mRNA transcript of an anti-apoptotic target gene,such as Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and Al.

In one aspect, the invention provides for a double-stranded ribonucleicacid (dsRNA) for inhibiting the expression of a bcl-2 gene in a cell.The dsRNA comprises at least two sequences that are complementary toeach other. The dsRNA comprises a sense strand comprising a firstsequence and an antisense strand comprising a second sequence. Theantisense strand comprises a nucleotide sequence which is substantiallycomplementary to at least part of an mRNA encoding bcl-2, and the regionof complementarity is less than 30 nucleotides in length. The dsRNA,upon contacting with a cell expressing the bcl-2, inhibits theexpression of the bcl-2 gene by at least 20%.

In one embodiment, the first sequence of the dsRNA is selected from thegroup consisting of SEQ ID NOs: (n) and the second sequence is selectedfrom the group consisting of SEQ ID NOs: (n+1), wherein n is an oddnumber in the range of 7-295. In another embodiment, n is an odd numberin the range of 7-209; in this embodiment, the dsRNA inhibits theexpression of the bcl-2 gene by at least 30%. In yet another embodiment,n is an odd number in the range of 7-169; in this embodiment, the dsRNAinhibits the expression of the bcl-2 gene by at least 40%. n can also bean odd number in the range of 7-129; in this embodiment, the dsRNAinhibits the expression of the bcl-2 gene by at least 50%. Preferably, nis an odd number in the range of 7-67; in this embodiment, the dsRNAinhibits the expression of the bcl-2 gene by at least 60%. Morepreferably, n is an odd number in the range of 7-31; in this embodiment,the dsRNA inhibits the expression of the bcl-2 gene by at least 70%.Most preferably, n is an odd number in the range of 7-11; in thisembodiment, the dsRNA inhibits the expression of the bcl-2 gene by atleast 80%.

In a another preferred embodiment, said dsRNA comprises at least onemodified nucleotide. Said modified nucleotide may be chosen from thegroup of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.Alternatively, said modified nucleotide may be chosen from the group of:a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.Preferably, the first sequence of said dsRNA is selected from the groupconsisting of SEQ ID NOs: (n) the said second sequence is selected fromthe group consisting of SEQ ID NOs: (n+1), wherein n is an odd number inthe range of 447-474. In another preferred embodiment, the said firstsequence is selected from the group consisting of SEQ ID NOs: (n) andsaid second sequence is selected from the group consisting of SEQ IDNOs: (n+1), wherein n is an odd number selected from the groupconsisting of: 475, 477, 471, 483, 485, 487, 489, 497, 499, 503.

In a second aspect, the invention provides for a cell comprising adouble-stranded ribonucleic acid (dsRNA) for inhibiting the expressionof a bcl-2 gene in a cell. The dsRNA comprises at least two sequencesthat are complementary to each other. The dsRNA comprises a sense strandcomprising a first sequence and an antisense strand comprising a secondsequence. The antisense strand comprises a nucleotide sequence which issubstantially complementary to at least part of an mRNA encoding bcl-2,and the region of complementarity is less than 30 nucleotides in length.The dsRNA, upon contacting with a cell expressing the bcl-2, inhibitsthe expression of the bcl-2 gene by at least 20%.

In one embodiment of said second aspect of the invention, the firstsequence of the dsRNA is selected from the group consisting of SEQ IDNOs: (n) and the second sequence is selected from the group consistingof SEQ ID NOs: (n+1), wherein n is an odd number in the range of 7-295.In another embodiment, n is an odd number in the range of 7-209; in thisembodiment, the dsRNA inhibits the expression of the bcl-2 gene by atleast 30%. In yet another embodiment, n is an odd number in the range of7-169; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 40%. n can also be an odd number in the range of7-129; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 50%. Preferably, n is an odd number in the rangeof 7-67; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 60%. More preferably, n is an odd number in therange of 7-31; in this embodiment, the dsRNA inhibits the expression ofthe bcl-2 gene by at least 70%. Most preferably, n is an odd number inthe range of 7-11; in this embodiment, the dsRNA inhibits the expressionof the bcl-2 gene by at least 80%.

In another preferred embodiment of said second aspect of the invention,said dsRNA comprises at least one modified nucleotide. Said modifiednucleotide may be chosen from the group of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group. Alternatively, said modified nucleotide may bechosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide. Preferably, the first sequence of said dsRNA is selectedfrom the group consisting of SEQ ID NOs: (n) the said second sequence isselected from the group consisting of SEQ ID NOs: (n+1), wherein n is anodd number in the range of 447-474. In another preferred embodiment, thesaid first sequence is selected from the group consisting of SEQ ID NOs:(n) and said second sequence is selected from the group consisting ofSEQ ID NOs: (n+1), wherein n is an odd number selected from the groupconsisting of: 475, 477, 471, 483, 485, 487, 489, 497, 499, 503.

In a third aspect, the present invention provides for a pharmaceuticalcomposition for inhibiting the expression of the bcl-2 gene in anorganism, comprising a dsRNA and a pharmaceutically acceptable carrier.The dsRNA comprises at least two sequences that are complementary toeach other, wherein a sense strand comprises a first sequence and anantisense strand comprises a second sequence. The antisense strandcomprises a region of complementarity which is substantiallycomplementary to at least a part of an mRNA encoding bcl-2. The regionof complementarity is less than 30 nucleotides in length. The dsRNA,upon contact with a cell expressing the bcl-2, inhibits expression ofthe bcl-2 gene by at least 20%.

In one embodiment of said third aspect of the invention, the firstsequence of the dsRNA is selected from the group consisting of SEQ IDNOs: (n) and the second sequence is selected from the group consistingof SEQ ID NOs: (n+1), wherein n is an odd number in the range of 7-295.In another embodiment, n is an odd number in the range of 7-209; in thisembodiment, the dsRNA inhibits the expression of the bcl-2 gene by atleast 30%. In yet another embodiment, n is an odd number in the range of7-169; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 40%. n can also be an odd number in the range of7-129; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 50%. Preferably, n is an odd number in the rangeof 7-67; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 60%. More preferably, n is an odd number in therange of 7-31; in this embodiment, the dsRNA inhibits the expression ofthe bcl-2 gene by at least 70%. Most preferably, n is an odd number inthe range of 7-11; in this embodiment, the dsRNA inhibits the expressionof the bcl-2 gene by at least 80%.

In a another preferred embodiment of said third aspect of the invention,said dsRNA comprises at least one modified nucleotide. Said modifiednucleotide may be chosen from the group of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group. Alternatively, said modified nucleotide may bechosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide. Preferably, the first sequence of said dsRNA is selectedfrom the group consisting of SEQ ID NOs: (n) the said second sequence isselected from the group consisting of SEQ ID NOs: (n+1), wherein n is anodd number in the range of 447-474. In another preferred embodiment, thesaid first sequence is selected from the group consisting of SEQ ID NOs:(n) and said second sequence is selected from the group consisting ofSEQ ID NOs: (n+1), wherein n is an odd number selected from the groupconsisting of: 475, 477, 471, 483, 485, 487, 489, 497, 499, 503.

In a fourth aspect of the invention, a method is provided for inhibitingthe expression of the bcl-2 gene in a cell, comprising the followingsteps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA comprises at least two sequences that        are complementary to each other. The dsRNA comprises a sense        strand comprising a first sequence and an antisense strand        comprising a second sequence. The antisense strand comprises a        region of complementarity which is substantially complementary        to at least a part of a mRNA encoding bcl-2, and wherein the        region of complementarity is less than 30 nucleotides in length        and wherein the dsRNA, upon contact with a cell expressing the        bcl-2, inhibits expression of the bcl-2 gene by at least 20%;        and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        bcl-2 gene, thereby inhibiting expression of the target gene in        the cell.

In one embodiment of said fourth aspect of the invention, the firstsequence of the dsRNA is selected from the group consisting of SEQ IDNOs: (n) and the second sequence is selected from the group consistingof SEQ ID NOs: (n+1), wherein n is an odd number in the range of 7-295.In another embodiment, n is an odd number in the range of 7-209; in thisembodiment, the dsRNA inhibits the expression of the bcl-2 gene by atleast 30%. In yet another embodiment, n is an odd number in the range of7-169; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 40%. n can also be an odd number in the range of7-129; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 50%. Preferably, n is an odd number in the rangeof 7-67; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 60%. More preferably, n is an odd number in therange of 7-31; in this embodiment, the dsRNA inhibits the expression ofthe bcl-2 gene by at least 70%. Most preferably, n is an odd number inthe range of 7-11; in this embodiment, the dsRNA inhibits the expressionof the bcl-2 gene by at least 80%.

In a another preferred embodiment of said fourth aspect of theinvention, said dsRNA comprises at least one modified nucleotide. Saidmodified nucleotide may be chosen from the group of: a 2′-O-methylmodified nucleotide, a nucleotide comprising a 5′-phosphorothioategroup, and a terminal nucleotide linked to a cholesteryl derivative ordodecanoic acid bisdecylamide group. Alternatively, said modifiednucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. Preferably, the firstsequence of said dsRNA is selected from the group consisting of SEQ IDNOs: (n) the said second sequence is selected from the group consistingof SEQ ID NOs: (n+1), wherein n is an odd number in the range of447-474. In another preferred embodiment, the said first sequence isselected from the group consisting of SEQ ID NOs: (n) and said secondsequence is selected from the group consisting of SEQ ID NOs: (n+1),wherein n is an odd number selected from the group consisting of: 475,477, 471, 483, 485, 487, 489, 497, 499, 503.

In a fifth aspect, the invention provides for a method of suppressinggrowth of a cancer cell, comprising contacting the cell with a dsRNA.The dsRNA comprises at least two sequences that are complementary toeach other. The dsRNA comprises a sense strand comprising a firstsequence and an antisense strand comprising a second sequence. Theantisense strand comprises a region of complementarity which issubstantially complementary to at least a part of an mRNA encodingbcl-2, and wherein the region of complementarity is less than 30nucleotides in length. The dsRNA, upon contact with a cell expressingthe bcl-2, inhibits expression of the bcl-2 gene by at least 20%.

In one embodiment of said fifth aspect of the invention, the firstsequence of the dsRNA is selected from the group consisting of SEQ IDNOs: (n) and the second sequence is selected from the group consistingof SEQ ID NOs: (n+1), wherein n is an odd number in the range of 7-295.In another embodiment, n is an odd number in the range of 7-209; in thisembodiment, the dsRNA inhibits the expression of the bcl-2 gene by atleast 30%. In yet another embodiment, n is an odd number in the range of7-169; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 40%. n can also be an odd number in the range of7-129; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 50%. Preferably, n is an odd number in the rangeof 7-67; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 60%. More preferably, n is an odd number in therange of 7-31; in this embodiment, the dsRNA inhibits the expression ofthe bcl-2 gene by at least 70%. Most preferably, n is an odd number inthe range of 7-11; in this embodiment, the dsRNA inhibits the expressionof the bcl-2 gene by at least 80%.

In a another preferred embodiment of said fifth aspect of the invention,said dsRNA comprises at least one modified nucleotide. Said modifiednucleotide may be chosen from the group of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group. Alternatively, said modified nucleotide may bechosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide. Preferably, the first sequence of said dsRNA is selectedfrom the group consisting of SEQ ID NOs: (n) the said second sequence isselected from the group consisting of SEQ ID NOs: (n+1), wherein n is anodd number in the range of 447-474. In another preferred embodiment, thesaid first sequence is selected from the group consisting of SEQ ID NOs:(n) and said second sequence is selected from the group consisting ofSEQ ID NOs: (n+1), wherein n is an odd number selected from the groupconsisting of: 475, 477, 471, 483, 485, 487, 489, 497, 499, 503.

In a sixth aspect of the invention, a method is provided for treating,preventing or managing cancer comprising administering to a patient inneed of such treatment, prevention or management a therapeutically orprophylactically effective amount of a dsRNA. The dsRNA comprises atleast two sequences that are complementary to each other. The dsRNAcomprises a sense strand comprising a first sequence and an antisensestrand comprising a second sequence. The antisense strand comprises aregion of complementarity which is substantially complementary to atleast a part of an mRNA encoding bcl-2. The region of complementarity isless than 30 nucleotides in length. The dsRNA, upon contact with a cellexpressing the bcl-2, inhibits expression of the bcl-2 gene by at least20%.

In one embodiment of said sixth aspect of the invention, the firstsequence of the dsRNA is selected from the group consisting of SEQ IDNOs: (n) and the second sequence is selected from the group consistingof SEQ ID NOs: (n+1), wherein n is an odd number in the range of 7-295.In another embodiment, n is an odd number in the range of 7-209; in thisembodiment, the dsRNA inhibits the expression of the bcl-2 gene by atleast 30%. In yet another embodiment, n is an odd number in the range of7-169; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 40%. n can also be an odd number in the range of7-129; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 50%. Preferably, n is an odd number in the rangeof 7-67; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 60%. More preferably, n is an odd number in therange of 7-31; in this embodiment, the dsRNA inhibits the expression ofthe bcl-2 gene by at least 70%. Most preferably, n is an odd number inthe range of 7-11; in this embodiment, the dsRNA inhibits the expressionof the bcl-2 gene by at least 80%.

In a another preferred embodiment of said sixth aspect of the invention,said dsRNA comprises at least one modified nucleotide. Said modifiednucleotide may be chosen from the group of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group. Alternatively, said modified nucleotide may bechosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide. Preferably, the first sequence of said dsRNA is selectedfrom the group consisting of SEQ ID NOs: (n) the said second sequence isselected from the group consisting of SEQ ID NOs: (n+1), wherein n is anodd number in the range of 447-474. In another preferred embodiment, thesaid first sequence is selected from the group consisting of SEQ ID NOs:(n) and said second sequence is selected from the group consisting ofSEQ ID NOs: (n+1), wherein n is an odd number selected from the groupconsisting of: 475, 477, 471, 483, 485, 487, 489, 497, 499, 503.

In a seventh aspect, the invention provides for a vector for inhibitingthe expression of a bcl-2 gene in a cell, comprising a regulatorysequence operably linked to a nucleotide sequence that encodes at leastone strand of a dsRNA. One of the strands of the dsRNA is substantiallycomplementary to at least a part of an mRNA encoding bcl-2 and the dsRNAis less than 50, preferably less than 30 base pairs in length. Uponintroduction of the vector into a cell expressing the bcl-2, andsubsequent expression of the at least one strand of the dsRNA from thevector inside the cell, the dsRNA inhibits the expression of the bcl-2gene by at least 20%.

In one embodiment of said seventh aspect of the invention, the firstsequence of the dsRNA is selected from the group consisting of SEQ IDNOs: (n) and the second sequence is selected from the group consistingof SEQ ID NOs: (n+1), wherein n is an odd number in the range of 7-295.In another embodiment, n is an odd number in the range of 7-209; in thisembodiment, the dsRNA inhibits the expression of the bcl-2 gene by atleast 30%. In yet another embodiment, n is an odd number in the range of7-169; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 40%. n can also be an odd number in the range of7-129; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 50%. Preferably, n is an odd number in the rangeof 7-67; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 60%. More preferably, n is an odd number in therange of 7-31; in this embodiment, the dsRNA inhibits the expression ofthe bcl-2 gene by at least 70%. Most preferably, n is an odd number inthe range of 7-11; in this embodiment, the dsRNA inhibits the expressionof the bcl-2 gene by at least 80%.

In an eighth embodiment, the invention provides for a cell comprising avector for inhibiting the expression of a bcl-2 gene in a cell. Thevector comprises a regulatory sequence operably linked to a nucleotidesequence that encodes at least one strand of a dsRNA. One of the strandsof the dsRNA is substantially complementary to at least a part of a mRNAencoding bcl-2 and (the dsRNA is less than 30 base pairs in length) Uponexpression of the at least one strand of a dsRNA from the vector insidethe cell, the dsRNA inhibits the expression of the bcl-2 gene in thecell by at least 20%.

In one embodiment of said eighth aspect of the invention, the firstsequence of the dsRNA is selected from the group consisting of SEQ IDNOs: (n) and the second sequence is selected from the group consistingof SEQ ID NOs: (n+1), wherein n is an odd number in the range of 7-295.In another embodiment, n is an odd number in the range of 7-209; in thisembodiment, the dsRNA inhibits the expression of the bcl-2 gene by atleast 30%. In yet another embodiment, n is an odd number in the range of7-169; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 40%. n can also be an odd number in the range of7-129; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 50%. Preferably, n is an odd number in the rangeof 7-67; in this embodiment, the dsRNA inhibits the expression of thebcl-2 gene by at least 60%. More preferably, n is an odd number in therange of 7-31; in this embodiment, the dsRNA inhibits the expression ofthe bcl-2 gene by at least 70%. Most preferably, n is an odd number inthe range of 7-11; in this embodiment, the dsRNA inhibits the expressionof the bcl-2 gene by at least 80%.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the apoptosis rate (percent) of human pancreatic YAP Ccancer cells, 120 hours after transfection with dsRNA 1 that iscomplementary to a first sequence of the human Bcl-2 gene.

FIG. 2 shows the apoptosis rate (percent) of YAP C cells, 120 hoursafter transfection with dsRNA 2 that is complementary to a firstsequence of the human Bcl-2 gene.

FIG. 3 shows the apoptosis rate (percent) of YAP C cells, 120 hoursafter transfection with dsRNA 3 that is complementary to a sequence ofthe neomycin resistance gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses double-stranded ribonucleic acid(dsRNA), as well as compositions and methods for inhibiting theexpression of a target gene in a cell using the dsRNA. The presentinvention also discloses compositions and methods for treating diseasesin organisms caused by the expression of an anti-apoptotic gene usingdsRNA. dsRNA directs the sequence-specific degradation of mRNA through aprocess known as RNA interference (RNAi). The process occurs in a widevariety of organisms, including mammals and other vertebrates.

The dsRNA of the invention comprises an RNA strand (the antisensestrand) having a region which is less than 30 nucleotides in length andis substantially complementary to at least part of an mRNA transcript ofan anti-apoptotic target gene, such as Bcl-2, Bcl-XL, Bcl-w, Mcl-1,and/or Al. The use of these dsRNAs enables the targeted degradation ofmRNAs of genes that are implicated in uncontrolled cell or tissuegrowth. Using cell-based assays, the present inventors have demonstratedthat very low dosages of these dsRNA can specifically and efficientlymediate RNAi, resulting in significant inhibition of expression of thetarget gene(s). Not only are lower dosages of dsRNA required as comparedto traditional antisense RNA, but dsRNA affects apoptosis to such anextent that there is a noticeable reduction in both tumor size andnumber of tumor cells. Thus, the present invention encompasses thesedsRNAs and compositions comprising dsRNA and their use for specificallysilencing genes whose protein products either inhibit or preventapoptosis in tumor cells. Moreover, the dsRNAs of the invention have noapparent effect on neighboring normal cells. Thus, the methods andcompositions of the present invention comprising these dsRNAs are usefulfor treating cellular proliferative and/or differentiation disorders,such as cancer.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression oftarget anti-apoptotic genes, as well as compositions and methods fortreating diseases and disorders caused by the expression of these genes.The pharmaceutical compositions of the present invention comprise adsRNA having an antisense strand comprising a region of complementaritywhich is less than 30 nucleotides in length and is substantiallycomplementary to at least part of an RNA transcript of an anti-apoptotictarget gene, together with a pharmaceutically acceptable carrier. Theanti-apoptotic gene may be a member of the Bcl-2 family, such as Bcl-2,Bcl-XL, Bcl-w, Mcl-1, and/or Al. The pharmaceutical composition maycomprise a combination of dsRNAs having regions complementary to aplurality of anti-apoptotic genes, for example a combination of Bcl-2,Bcl-XL, Bcl-w, Mcl-1 and/or Al. Since many types of tumor cells areknown to express multiple anti-apoptotic genes, compositions comprisinga combination of dsRNAs are particularly effective at inhibiting thedevelopment and/or growth of tumor cells.

Accordingly, certain aspects of the present invention relate topharmaceutical compositions comprising the dsRNA of the presentinvention together with a pharmaceutically acceptable carrier, methodsof using the compositions to inhibit expression of a targetanti-apoptotic gene, and methods of using the pharmaceuticalcompositions to treat diseases caused by expression of at least one ofthese anti-apoptotic genes.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, and uracil may be replacedby other moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of thepresent invention by a nucleotide containing, for example, inosine.Sequences comprising such replacement moieties are embodiments of thepresent invention.

As used herein, “target gene” refers to a section of a DNA strand of adouble-stranded DNA that is complementary to a section of a DNA strand,including all transcribed regions, that serves as a matrix fortranscription. A target gene is a gene whose expression is to beselectively inhibited or silenced through RNA interference. As usedherein, the term “target gene” specifically encompasses any cellulargene or gene fragment whose expression or activity is associated withthe inhibition or prevention of apoptosis. For example, the target genemay be a gene from the Bcl-2 gene family, such as Bcl-2, Bcl-XL, Bcl-w,Mcl-1, and/or Al.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a target gene, including mRNA that is a product of RNA processing ofa primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butpreferably not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the present invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding bcl-2). For example, a polynucleotide iscomplementary to at least a part of a bcl-2 mRNA if the sequence issubstantially complementary to a non-interrupted portion of a mRNAencoding bcl-2.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to aribonucleic acid molecule, or complex of ribonucleic acid molecules,having a duplex structure comprising two anti-parallel and substantiallycomplementary, as defined above, nucleic acid strands. The two strandsforming the duplex structure may be different portions of one larger RNAmolecule, or they may be separate RNA molecules. Where the two strandsare part of one larger molecule, and therefore are connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting RNA chain is referred to as a “hairpin loop”. Where thetwo strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker”. The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA. In addition to the duplex structure, a dsRNA may compriseone or more nucleotide overhangs.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are preferably in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as theyrefer to a target gene, herein refer to the at least partial suppressionof the expression of the target gene, as manifested by a reduction ofthe amount of mRNA transcribed from the target gene which may beisolated from a first cell or group of cells in which the target gene istranscribed and which has or have been treated such that the expressionof the target gene is inhibited, as compared to a second cell or groupof cells substantially identical to the first cell or group of cells butwhich has or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to target genetranscription, e.g. the amount of protein encoded by the target genewhich is secreted by a cell, or the number of cells displaying a certainphenotype, e.g apoptosis. In principle, target gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given siRNA inhibitsthe expression of the target gene by a certain degree and therefore isencompassed by the instant invention, the KB-GFP-BCL2 and assay ofExample 1 herein below shall serve as such reference.

For example, in certain instances, expression of the target gene issuppressed by at least about 20%, 25%, 35%, or 50% by administration ofthe double-stranded oligonucleotide of the invention. In a preferredembodiment, the target gene is suppressed by at least about 60%, 70%, or80% by administration of the double-stranded oligonucleotide of theinvention. In a more preferred embodiment, the target gene is suppressedby at least about 85%, 90%, or 95% by administration of thedouble-stranded oligonucleotide of the invention. In a most preferredembodiment, the target gene is suppressed by at least about 98%, 99% ormore by administration of the double-stranded oligonucleotide of theinvention.

As used herein, the term “treatment” refers to the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disorder, e.g., a disease or condition, asymptom of disease, or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the disease, the symptoms of disease, or thepredisposition toward disease. A “patient” may be a human, but can alsobe a non-human animal.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management of adisease or condition, e.g. cancer. The specific amount that istherapeutically effective can be readily determined by ordinary medicalpractitioner, and may vary depending on factors known in the art, suchas, e.g. the type of cancer, the patient's history and age, the stage ofcancer, the administration of other anti-cancer agents, includingradiation therapy.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA)

In one embodiment, the invention relates to a double-strandedribonucleic acid (dsRNA) for inhibiting the expression of a target genein a cell, wherein the dsRNA comprises an antisense strand comprising aregion of complementarity which is complementary to at least a part ofan mRNA formed in the expression of the target gene, and wherein theregion of complementarity is less than 30 nucleotides in length andwherein said dsRNA, upon contact with a cell expressing said targetgene, inhibits the expression of said target gene by at least 20%. ThedsRNA comprises two RNA strands that are sufficiently complementary tohybridize to form a duplex structure. One strand of the dsRNA (theantisense strand) comprises a region of complementarity that issubstantially complementary, and preferably fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of the target gene, the other strand (the sense strand)comprises a region which is complementary to the antisense strand, suchthat the two strands hybridize and form a duplex structure when combinedunder suitable conditions. Preferably, the duplex structure is between15 and 30, more preferably between 18 and 25, yet more preferablybetween 19 and 24, and most preferably between 21 and 23 base pairs inlength. Similarly, the region of complementarity to the target sequenceis between 15 and 30, more preferably between 18 and 25, yet morepreferably between 19 and 24, and most preferably between 21 and 23nucleotides in length. The dsRNA of the present invention may furthercomprise one or more single-stranded nucleotide overhang(s). The dsRNAcan be synthesized by standard methods known in the art as furtherdiscussed below, e.g., by use of an automated DNA synthesizer, such asare commercially available from, for example, Biosearch, AppliedBiosystems, Inc. In a preferred embodiment, the target gene is a memberof the Bcl-2 family, e.g., Bcl-2, Bcl-XL, Bcl-w, Mcl-1 or AI. In aparticularly preferred embodiment, the target gene is Bcl-2. In specificembodiments, the antisense strand of the dsRNA comprises the sequenceset forth in SEQ ID NO:2 and the sense strand comprises the sequence setforth in SEQ ID NO:1; or the antisense strand of the dsRNA comprises thesequence set forth in SEQ ID NO:4 and the sense strand comprises thesequence set forth in SEQ ID NO:3.

In further embodiments, the dsRNA comprises at least one nucleotidesequence selected from the groups of SEQ ID NOs: 7-296, 7-210, 7-170,7-130, 7-68, 7-32, or 7-12. In other embodiments, the dsRNA comprises atleast two sequences selected from these groups, wherein one of the atleast two sequences is complementary to another of the at least twosequences, and one of the at least two sequences is substantiallycomplementary to a sequence of an mRNA generated in the expression of abcl-2 gene. Preferably, the dsRNA comprises two oligonucleotides,wherein one oligonucleotide is described by SEQ ID NO: (n) and thesecond oligonucleotide is described SEQ ID NO: (n+1), n being an oddnumber in the range of 7-295, for example in the range of 7-209, 7-169,7-129, 7-67, 7-31, or 7-11.

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences SEQ ID NOs: 7-296, the dsRNAs of the presentinvention comprise at least one strand of a length of minimally 21 nt.It can be reasonably expected that shorter dsRNAs comprising one of thesequences of SEQ ID NOs: 7-296 minus only a few nucleotides on one orboth ends may be similarly effective as compared to the dsRNAs describedabove. Hence, dsRNAs comprising a partial sequence of at least 15, 16,17, 18, 19, 20, or more contiguous nucleotides from one of the sequencesof SEQ ID NOs: 7-296, and differing in their ability to inhibit theexpression of a bcl-2 gene in a FACS assay as described herein below bynot more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNAcomprising the full sequence, are contemplated by the present invention.

The dsRNA of the present invention can contain one or more mismatches tothe target sequence. In a preferred embodiment, the dsRNA of the presentinvention contains no more than 3 mismatches. If the antisense strand ofthe dsRNA contains mismatches to a target sequence, it is preferablethat the area of mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of a bcl-2 gene, the dsRNA preferably does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the present invention can be used to determine whethera dsRNA containing a mismatch to a target sequence is effective ininhibiting the expression of the target gene. Consideration of theefficacy of dsRNAs with mismatches in inhibiting expression of thetarget gene is important, especially if the particular region ofcomplementarity in the target gene is known to have polymorphic sequencevariation within the population.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, preferably 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. Moreover, thepresent inventors have discovered that the presence of only onenucleotide overhang strengthens the interference activity of the dsRNA,without affecting its overall stability. dsRNA having only one overhanghas proven particularly stable and effective in vivo, as well as in avariety of cells, cell culture mediums, blood, and serum. Preferably,the single-stranded overhang is located at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, preferably located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Preferably, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of the present invention may be synthesizedand/or modified by methods well established in the art, such as thosedescribed in “Current protocols in nucleic acid chemistry”, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, whichis hereby incorporated herein by reference. Chemical modifications mayinclude, but are not limited to 2′ modifications, introduction ofnon-natural bases, covalent attachment to a ligand, and replacement ofphosphate linkages with thiophosphate linkages. In this embodiment, theintegrity of the duplex structure is strengthened by at least one, andpreferably two, chemical linkages. Chemical linking may be achieved byany of a variety of well-known techniques, for example by introducingcovalent, ionic or hydrogen bonds; hydrophobic interactions, van derWaals or stacking interactions; by means of metal-ion coordination, orthrough use of purine analogues. Preferably, the chemical groups thatcan be used to modify the dsRNA include, without limitation, methyleneblue; bifunctional groups, preferably bis-(2-chloroethyl)amine;N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. Inone preferred embodiment, the linker is a hexa-ethylene glycol linker.In this case, the dsRNA are produced by solid phase synthesis and thehexa-ethylene glycol linker is incorporated according to standardmethods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996)35:14665-14670). In a particular embodiment, the 5′-end of the antisensestrand and the 3′-end of the sense strand are chemically linked via ahexaethylene glycol linker. In another embodiment, at least onenucleotide of the dsRNA comprises a phosphorothioate orphosphorodithioate groups. The chemical bond at the ends of the dsRNA ispreferably formed by triple-helix bonds.

In certain embodiments, a chemical bond may be formed by means of one orseveral bonding groups, wherein such bonding groups are preferablypoly-(oxyphosphinicooxy-1,3-propandiol)- and/or polyethylene glycolchains. In other embodiments, a chemical bond may also be formed bymeans of purine analogs introduced into the double-stranded structureinstead of purines. In further embodiments, a chemical bond may beformed by azabenzene units introduced into the double-strandedstructure. In still further embodiments, a chemical bond may be formedby branched nucleotide analogs instead of nucleotides introduced intothe double-stranded structure. In certain embodiments, a chemical bondmay be induced by ultraviolet light.

In yet another embodiment, the nucleotides at one or both of the twosingle strands may be modified to prevent or inhibit the activation ofcellular enzymes, such as, for example, without limitation, certainnucleases. Techniques for inhibiting the activation of cellular enzymesare known in the art including, but not limited to, 2′-aminomodifications, 2′-amino sugar modifications, 2′-F sugar modifications,2′-F modifications, 2′-alkyl sugar modifications, uncharged backbonemodifications, morpholino modifications, 2′-O-methyl modifications, andphosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, atleast one 2′-hydroxyl group of the nucleotides on a dsRNA is replaced bya chemical group, preferably by a 2′-amino or a 2′-methyl group. Also,at least one nucleotide may be modified to form a locked nucleotide.Such locked nucleotide contains a methylene bridge that connects the2′-oxygen of ribose with the 4′-carbon of ribose. Oligonucleotidescontaining the locked nucleotide are described in Koshkin, A. A., etal., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al.,Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a lockednucleotide into an oligonucleotide improves the affinity forcomplementary sequences and increases the melting temperature by severaldegrees (Braasch, D. A. and D. R. Corey, Chem. Biol. (2001), 8:1-7).

Conjugating a ligand to a dsRNA can enhance its cellular absorption. Incertain instances, a hydrophobic ligand is conjugated to the dsRNA tofacilitate direct permeation of the cellular membrane. Alternatively,the ligand conjugated to the dsRNA is a substrate for receptor-mediatedendocytosis. These approaches have been used to facilitate cellpermeation of antisense oligonucleotides. For example, cholesterol hasbeen conjugated to various antisense oligonucleotides resulting incompounds that are substantially more active compared to theirnon-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid DrugDevelopment 2002, 12, 103. Other lipophilic compounds that have beenconjugated to oligonucleotides include 1-pyrene butyric acid,1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand forreceptor-mediated endocytosis is folic acid. Folic acid enters the cellby folate-receptor-mediated endocytosis. dsRNA compounds bearing folicacid would be efficiently transported into the cell via thefolate-receptor-mediated endocytosis. Li and coworkers report thatattachment of folic acid to the 3′-terminus of an oligonucleotideresulted in an 8-fold increase in cellular uptake of theoligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998,15, 1540. Other ligands that have been conjugated to oligonucleotidesinclude polyethylene glycols, carbohydrate clusters, cross-linkingagents, porphyrin conjugates, and delivery peptides.

In certain instances, conjugation of a cationic ligand tooligonucleotides often results in improved resistance to nucleases.Representative examples of cationic ligands are propylammonium anddimethylpropylammonium. Interestingly, antisense oligonucleotides werereported to retain their high binding affinity to mRNA when the cationicligand was dispersed throughout the oligonucleotide. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103 and referencestherein.

The ligand-conjugated dsRNA of the invention may be synthesized by theuse of a dsRNA that bears a pendant reactive functionality, such as thatderived from the attachment of a linking molecule onto the dsRNA. Thisreactive oligonucleotide may be reacted directly withcommercially-available ligands, ligands that are synthesized bearing anyof a variety of protecting groups, or ligands that have a linking moietyattached thereto. The methods of the present invention facilitate thesynthesis of ligand-conjugated dsRNA by the use of, in some preferredembodiments, nucleoside monomers that have been appropriately conjugatedwith ligands and that may further be attached to a solid-supportmaterial. Such ligand-nucleoside conjugates, optionally attached to asolid-support material, are prepared according to some preferredembodiments of the methods of the present invention via reaction of aselected serum-binding ligand with a linking moiety located on the 5′position of a nucleoside or oligonucleotide. In certain instances, andsRNA bearing an aralkyl ligand attached to the 3′-terminus of the dsRNAis prepared by first covalently attaching a monomer building block to acontrolled-pore-glass support via a long-chain aminoalkyl group. Then,nucleotides are bonded via standard solid-phase synthesis techniques tothe monomer building-block bound to the solid support. The monomerbuilding block may be a nucleoside or other organic compound that iscompatible with solid-phase synthesis.

The dsRNA used in the conjugates of the present invention may beconveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. patents: U.S. Pat.Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugatedoligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for thepreparation of oligonucleotides having chiral phosphorus linkages; U.S.Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides havingmodified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modifiedoligonucleotides and the preparation thereof through reductive coupling;U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the3-deazapurine ring system and methods of synthesis thereof; U.S. Pat.No. 5,459,255, drawn to modified nucleobases based on N-2 substitutedpurines; U.S. Pat. No. 5,521,302, drawn to processes for preparingoligonucleotides having chiral phosphorus linkages; U.S. Pat. No.5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No.5,571,902, drawn to methods and materials for the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides havingalkylthio groups, wherein such groups may be used as linkers to othermoieties attached at any of a variety of positions of the nucleoside;U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides havingphosphorothioate linkages of high chiral purity; U.S. Pat. No.5,506,351, drawn to processes for the preparation of 2′-O-alkylguanosine and related compounds, including 2,6-diaminopurine compounds;U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotideshaving 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No.5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs;U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modifiedoligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawnto, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.

In the ligand-conjugated dsRNA and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide.Oligonucleotide conjugates bearing a variety of molecules such assteroids, vitamins, lipids and reporter molecules, has previously beendescribed (see Manoharan et al., PCT Application WO 93/07883). In apreferred embodiment, the oligonucleotides or linked nucleosides of thepresent invention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl,2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of anoligonucleotide confers enhanced hybridization properties to theoligonucleotide. Further, oligonucleotides containing phosphorothioatebackbones have enhanced nuclease stability. Thus, functionalized, linkednucleosides of the invention can be augmented to include either or botha phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group.

In some preferred embodiments, functionalized nucleoside sequences ofthe invention possessing an amino group at the 5′-terminus are preparedusing a DNA synthesizer, and then reacted with an active esterderivative of a selected ligand. Active ester derivatives are well knownto those skilled in the art. Representative active esters includeN-hydrosuccinimide esters, tetrafluorophenolic esters,pentafluorophenolic esters and pentachlorophenolic esters. The reactionof the amino group and the active ester produces an oligonucleotide inwhich the selected ligand is attached to the 5′-position through alinking group. The amino group at the 5′-terminus can be preparedutilizing a 5′-Amino-Modifier C6 reagent. In a preferred embodiment,ligand molecules may be conjugated to oligonucleotides at the5′-position by the use of a ligand-nucleoside phosphoramidite whereinthe ligand is linked to the 5′-hydroxy group directly or indirectly viaa linker. Such ligand-nucleoside phosphoramidites are typically used atthe end of an automated synthesis procedure to provide aligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.

In one preferred embodiment of the methods of the invention, thepreparation of ligand conjugated oligonucleotides commences with theselection of appropriate precursor molecules upon which to construct theligand molecule. Typically, the precursor is an appropriately-protectedderivative of the commonly-used nucleosides. For example, the syntheticprecursors for the synthesis of the ligand-conjugated oligonucleotidesof the present invention include, but are not limited to,2′-aminoalkoxy-5′-ODMT-nucleosides,2′-6-aminoalkylamino-5′-ODMT-nucleosides,5′-6-aminoalkoxy-2′-deoxy-nucleosides,5′-6-aminoalkoxy-2-protected-nucleosides,3′-6-aminoalkoxy-5′-ODMT-nucleosides, and3′-aminoalkylamino-5′-ODMT-nucleosides that may be protected in thenucleobase portion of the molecule. Methods for the synthesis of suchamino-linked protected nucleoside precursors are known to those ofordinary skill in the art.

In many cases, protecting groups are used during the preparation of thecompounds of the invention. As used herein, the term “protected” meansthat the indicated moiety has a protecting group appended thereon. Insome preferred embodiments of the invention, compounds contain one ormore protecting groups. A wide variety of protecting groups can beemployed in the methods of the invention. In general, protecting groupsrender chemical functionalities inert to specific reaction conditions,and can be appended to and removed from such functionalities in amolecule without substantially damaging the remainder of the molecule.

The present inventors have identified certain sequence motifs that areparticularly prone to degradative attack by endonucleases, seeco-pending and co-owned U.S. 60/574,744 and PCT/US2005/018931, both ofwhich are hereby incorporated by reference in their entirety.Consequently, the protecting groups are preferably introduced withinthese sites of preferential degradation. For example, in certainembodiments, all the pyrimidines of an iRNA agent carry a 2′-protectinggroup, and the iRNA agent therefore has enhanced resistance toendonucleases. Enhanced nuclease resistance can also be achieved byprotecting the 5′ nucleotide, resulting, for example, in at least one5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein the uridine is a2′-protected nucleotide; at least one 5′-uridine-guanine-3′ (5′-UG-3′)dinucleotide, wherein the 5′-uridine is a 2′-protected nucleotide; atleast one 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the5′-cytidine is a 2′-protected nucleotide; at least one5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-protected nucleotide; or at least one 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-protectednucleotide. The iRNA agent can include at least 2, at least 3, at least4 or at least 5 of such protected dinucleotides. Most preferably, the5′-most pyrimidines in all occurrences of the sequence motifs5′-UA-3′,5′-CA-3′,5′-UU-3′, and 5′-UG-3′ are 2′-protected nucleotides.Alternatively, all pyrimidines of the sense strand of an iRNA agentcarry a 2′-protecting group, and all occurrences of the sequence motifs5′-UA-3′,5′-CA-3′,5′-UU-3′, and 5′-UG-3′ in the antisense strand are2′-protected nucleotides.

Representative hydroxyl protecting groups, for example, are disclosed byBeaucage et al. (Tetrahedron, 1992, 48:2223-2311). Further hydroxylprotecting groups, as well as other representative protecting groups,are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, andOligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed.,IRL Press, N.Y, 1991.

Examples of hydroxyl protecting groups include, but are not limited to,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl,diphenylmethyl, p,p′-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate,chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate,p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.

Amino-protecting groups stable to acid treatment are selectively removedwith base treatment, and are used to make reactive amino groupsselectively available for substitution. Examples of such groups are theFmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J.Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p. 1) andvarious substituted sulfonylethyl carbamates exemplified by the Nscgroup (Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart andTesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).

Additional amino-protecting groups include, but are not limited to,carbamate protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamideprotecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclicimide protecting groups, such as phthalimido and dithiasuccinoyl.Equivalents of these amino-protecting groups are also encompassed by thecompounds and methods of the present invention.

Many solid supports are commercially available and one of ordinary skillin the art can readily select a solid support to be used in thesolid-phase synthesis steps. In certain embodiments, a universal supportis used. A universal support allows for preparation of oligonucleotideshaving unusual or modified nucleotides located at the 3′-terminus of theoligonucleotide. Universal Support 500 and Universal Support II areuniversal supports that are commercially available from Glen Research,22825 Davis Drive, Sterling, Va. For further details about universalsupports see Scott et al., Innovations and Perspectives in solid-phaseSynthesis, 3rd International Symposium, 1994, Ed. Roger Epton, MayflowerWorldwide, 115-124]; Azhayev, A. V. Tetrahedron 1999, 55, 787-800; andAzhayev and Antopolsky Tetrahedron 2001, 57, 4977-4986. In addition, ithas been reported that the oligonucleotide can be cleaved from theuniversal support under milder reaction conditions when oligonucleotideis bonded to the solid support via a syn-1,2-acetoxyphosphate groupwhich more readily undergoes basic hydrolysis. See Guzaev, A. I.;Manoharan, M. J. Am. Chem. Soc. 2003, 125, 2380.

The nucleosides are linked by phosphorus-containing ornon-phosphorus-containing covalent internucleoside linkages. For thepurposes of identification, such conjugated nucleosides can becharacterized as ligand-bearing nucleosides or ligand-nucleosideconjugates. The linked nucleosides having an aralkyl ligand conjugatedto a nucleoside within their sequence will demonstrate enhanced dsRNAactivity when compared to like dsRNA compounds that are not conjugated.

The aralkyl-ligand-conjugated oligonucleotides of the present inventionalso include conjugates of oligonucleotides and linked nucleosideswherein the ligand is attached directly to the nucleoside or nucleotidewithout the intermediacy of a linker group. The ligand may preferably beattached, via linking groups, at a carboxyl, amino or oxo group of theligand. Typical linking groups may be ester, amide or carbamate groups.

Specific examples of preferred modified oligonucleotides envisioned foruse in the ligand-conjugated oligonucleotides of the present inventioninclude oligonucleotides containing modified backbones or non-naturalinternucleoside linkages. As defined here, oligonucleotides havingmodified backbones or internucleoside linkages include those that retaina phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of the invention,modified oligonucleotides that do not have a phosphorus atom in theirintersugar backbone can also be considered to be oligonucleosides.

Specific oligonucleotide chemical modifications are described below. Itis not necessary for all positions in a given compound to be uniformlymodified. Conversely, more than one modifications may be incorporated ina single dsRNA compound or even in a single nucleotide thereof.

Preferred modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Representative United States patents relating to the preparation of theabove phosphorus-atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is hereinincorporated by reference.

Preferred modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents relating to the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleoside units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigonucleotide, an oligonucleotide mimetic, that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide-containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to atoms of the amide portion of the backbone.Representative United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found in Nielsen etal., Science, 1991, 254, 1497.

Some preferred embodiments of the present invention employoligonucleotides with phosphorothioate linkages and oligonucleosideswith heteroatom backbones, and in particular —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂—, and—O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone isrepresented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No.5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are oligonucleotides having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

The oligonucleotides employed in the ligand-conjugated oligonucleotidesof the present invention may additionally or alternatively comprisenucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases, such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligonucleotides of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Id., pages 276-278) and are presentlypreferred base substitutions, even more particularly when combined with2′-methoxyethyl sugar modifications.

Representative United States patents relating to the preparation ofcertain of the above-noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; and 5,808,027; all of which are herebyincorporated by reference.

In certain embodiments, the oligonucleotides employed in theligand-conjugated oligonucleotides of the present invention mayadditionally or alternatively comprise one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl, O-, S-, or N-alkenyl, or O,S- or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. apreferred modification includes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim.Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in U.S. Pat. No. 6,127,533,filed on Jan. 30, 1998, the contents of which are incorporated byreference.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides.

As used herein, the term “sugar substituent group” or “2′-substituentgroup” includes groups attached to the 2′-position of the ribofuranosylmoiety with or without an oxygen atom. Sugar substituent groups include,but are not limited to, fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole andpolyethers of the formula (O-alkyl)_(m), wherein m is 1 to about 10.Preferred among these polyethers are linear and cyclic polyethyleneglycols (PEGs), and (PEG)-containing groups, such as crown ethers andthose which are disclosed by Ouchi et al. (Drug Design and Discovery1992, 9:93); Ravasio et al. (J. Org. Chem. 1991, 56:4329); and Delgardoet. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992,9:249), each of which is hereby incorporated by reference in itsentirety. Further sugar modifications are disclosed by Cook (Anti-CancerDrug Design, 1991, 6:585-607). Fluoro, O-alkyl, O-alkylamino, O-alkylimidazole, O-alkylaminoalkyl, and alkyl amino substitution is describedin U.S. Pat. No. 6,166,197, entitled “Oligomeric Compounds havingPyrimidine Nucleotide(s) with 2′ and 5′ Substitutions,” herebyincorporated by reference in its entirety.

Additional sugar substituent groups amenable to the present inventioninclude 2′-SR and 2′-NR₂ groups, wherein each R is, independently,hydrogen, a protecting group or substituted or unsubstituted alkyl,alkenyl, or alkynyl. 2′-SR Nucleosides are disclosed in U.S. Pat. No.5,670,633, hereby incorporated by reference in its entirety. Theincorporation of 2′-SR monomer synthons is disclosed by Hamm et al. (J.Org. Chem., 1997, 62:3415-3420). 2′-NR nucleosides are disclosed byGoettingen, M., J. Org. Chem., 1996, 61, 6273-6281; and Polushin et al.,Tetrahedron Lett., 1996, 37, 3227-3230. Further representative2′-substituent groups amenable to the present invention include thosehaving one of formula I or II:

wherein,

E is C₁-C₁₀ alkyl, N(Q₃)(Q₄) or N═C (Q₃)(Q₄); each Q₃ and Q₄ is,independently, H, C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protectinggroup, a tethered or untethered conjugate group, a linker to a solidsupport; or Q₃ and Q₄, together, form a nitrogen protecting group or aring structure optionally including at least one additional heteroatomselected from N and O;

q₁ is an integer from 1 to 10;

q₂ is an integer from 1 to 10;

q₃ is 0 or 1;

q₄ is 0, 1 or 2;

each Z₁, Z₂ and Z₃ is, independently, C₄-C₇ cycloalkyl, C₅-C₁₄ aryl orC₃-C₁₅ heterocyclyl, wherein the heteroatom in said heterocyclyl groupis selected from oxygen, nitrogen and sulfur;

Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is, independently, H, C₁-C₈ alkyl,C₁-C₈ haloalkyl, C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H orC₁-C₈ alkyl; and

Z₅ is C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,C₆-C₁₄ aryl, N(Q₃)(Q₄), OQ₃, halo, SQ₃ or CN.

Representative 2′-O-sugar substituent groups of formula I are disclosedin U.S. Pat. No. 6,172,209, entitled “Capped 2′-OxyethoxyOligonucleotides,” hereby incorporated by reference in its entirety.Representative cyclic 2′-O-sugar substituent groups of formula II aredisclosed in U.S. Pat. No. 6,271,358, entitled “RNA Targeted 2′-ModifiedOligonucleotides that are Conformationally Preorganized,” herebyincorporated by reference in its entirety.

Sugars having O-substitutions on the ribosyl ring are also amenable tothe present invention. Representative substitutions for ring O include,but are not limited to, S, CH₂, CHF, and CF₂. See, e.g., Secrist et al.,Abstract 21, Program & Abstracts, Tenth International Roundtable,Nucleosides, Nucleotides and their Biological Applications, Park City,Utah, Sep. 16-20, 1992.

Oligonucleotides may also have sugar mimetics, such as cyclobutylmoieties, in place of the pentofuranosyl sugar. Representative UnitedStates patents relating to the preparation of such modified sugarsinclude, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920; and5,859,221, all of which are hereby incorporated by reference.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide. For example, one additional modification of theligand-conjugated oligonucleotides of the present invention involveschemically linking to the oligonucleotide one or more additionalnon-ligand moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide. Such moietiesinclude but are not limited to lipid moieties, such as a cholesterolmoiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553),cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053),a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y.Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov etal., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75,49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative United States patents relating to the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941, each of whichis herein incorporated by reference.

The present invention also includes compositions employingoligonucleotides that are substantially chirally pure with regard toparticular positions within the oligonucleotides. Examples ofsubstantially chirally pure oligonucleotides include, but are notlimited to, those having phosphorothioate linkages that are at least 75%Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those havingsubstantially chirally pure (Sp or Rp) alkylphosphonate, phosphoramidateor phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and5,521,302).

In certain instances, the oligonucleotide may be modified by anon-ligand group. A number of non-ligand molecules have been conjugatedto oligonucleotides in order to enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide, and proceduresfor performing such conjugations are available in the scientificliterature. Such non-ligand moieties have included lipid moieties, suchas cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994,4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann.N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem.Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov etal., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993,75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such oligonucleotide conjugates have beenlisted above. Typical conjugation protocols involve the synthesis ofoligonucleotides bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction may be performed either with the oligonucleotidestill bound to the solid support or following cleavage of theoligonucleotide in solution phase. Purification of the oligonucleotideconjugate by HPLC typically affords the pure conjugate.

Alternatively, the molecule being conjugated may be converted into abuilding block, such as a phosphoramidite, via an alcohol group presentin the molecule or by attachment of a linker bearing an alcohol groupthat may be phosphitylated.

Importantly, each of these approaches may be used for the synthesis ofligand conjugated oligonucleotides. Aminolinked oligonucleotides may becoupled directly with ligand via the use of coupling reagents orfollowing activation of the ligand as an NHS or pentfluorophenolateester. Ligand phosphoramidites may be synthesized via the attachment ofan aminohexanol linker to one of the carboxyl groups followed byphosphitylation of the terminal alcohol functionality. Other linkers,such as cysteamine, may also be utilized for conjugation to achloroacetyl linker present on a synthesized oligonucleotide.

III. PHARMACEUTICAL COMPOSITIONS COMPRISING dsRNA

In one embodiment, the invention relates to a pharmaceutical compositioncomprising a dsRNA, as described in the preceding section, and apharmaceutically acceptable carrier, as described below. Thepharmaceutical composition comprising the dsRNA is useful for treating adisease or disorder associated with the expression or activity of ananti-apoptotic gene.

In another embodiment, the invention relates to a pharmaceuticalcomposition comprising at least two dsRNAs, designed to target differentanti-apoptotic genes, and a pharmaceutically acceptable carrier. Theanti-apoptotic genes may be members of the Bcl-2 family, such as Bcl-2,Bcl-XL, Bcl-w, Mcl-1, and/or Al. Due of the targeting of mRNA ofmultiple anti-apoptotic genes, pharmaceutical compositions comprising aplurality of dsRNAs may provide improved efficiency of treatment ascompared to compositions comprising a single dsRNA, at least in tumorcells expressing these multiple genes. In this embodiment, theindividual dsRNAs are prepared as described in the preceding section,which is incorporated by reference herein. One dsRNA can have anucleotide sequence which is substantially complementary to at leastpart of one anti-apoptotic gene; additional dsRNAs are prepared, each ofwhich has a nucleotide sequence that is substantially complementary topart of a different anti-apoptotic gene. For example, one dsRNA may havea nucleotide sequence that is substantially complementary to a Bcl-2gene, another dsRNA may have a nucleotide sequence that is substantiallycomplementary to a Bcl-xL gene, and yet another dsRNA may have anucleotide sequence that is substantially complementary to a Bcl-w gene.The multiple dsRNAs may be combined in the same pharmaceuticalcomposition, or formulated separately. If formulated individually, thecompositions containing the separate dsRNAs may comprise the same ordifferent carriers, and may be administered using the same or differentroutes of administration. Moreover, the pharmaceutical compositionscomprising the individual dsRNAs may be administered substantiallysimultaneously, sequentially, or at preset intervals throughout the dayor treatment period. Although the foregoing description relates totarget genes from the Bcl-2 family, the present invention encompassesany gene or combination of genes that have an inhibitory or preventiveeffect on apoptosis.

The pharmaceutical compositions of the present invention areadministered in dosages sufficient to inhibit expression of the targetgene. The present inventors have found that, because of their improvedefficiency, compositions comprising the dsRNA of the invention can beadministered at surprisingly low dosages. A maximum dosage of 5 mg dsRNAper kilogram body weight of recipient per day is sufficient to inhibitor completely suppress expression of the target gene.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0milligrams per kilogram body weight of the recipient per day, preferablyin the range of 0.1 to 200 micrograms per kilogram body weight per day,more preferably in the range of 0.1 to 100 micrograms per kilogram bodyweight per day, even more preferably in the range of 1.0 to 50micrograms per kilogram body weight per day, and most preferably in therange of 1.0 to 25 micrograms per kilogram body weight per day. Thepharmaceutical composition may be administered once daily, or the dsRNAmay be administered as two, three, four, five, six or more sub-doses atappropriate intervals throughout the day. In that case, the dsRNAcontained in each sub-dose must be correspondingly smaller in order toachieve the total daily dosage. The dosage unit can also be compoundedfor delivery over several days, e.g., using a conventional sustainedrelease formulation which provides sustained release of the dsRNA over aseveral day period. Sustained release formulations are well known in theart. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. For example, mouse models areavailable for hematopoietic malignancies such as leukemias, lymphomasand acute myelogenous leukemia. The MMHCC (Mouse models of Human CancerConsortium) web page (emice.nci.nih.gov), sponsored by the NationalCancer Institute, provides disease-site-specific compendium of knowncancer models, and has links to the searchable Cancer Models Database(cancermodels.nci.nih.gov), as well as the NCI-MMHCC mouse repository.Examples of the genetic tools that are currently available for themodeling of leukemia and lymphomas in mice, and which are useful inpracticing the present invention, are described in the followingreferences: Maru, Y., Int. J. Hematol. (2001) 73:308-322; Pandolfi, P.P., Oncogene (2001) 20:5726-5735; Pollock, J. L., et al., Curr. Opin.Hematol. (2001) 8:206-211; Rego, E. M., et al., Semin. in Hemat. (2001)38:4-70; Shannon, K. M., et al. (2001) Modeling myeloid leukemia tumorssuppressor gene inactivation in the mouse, Semin. Cancer Biol. 11,191-200; Van Etten, R. A., (2001) Curr. Opin. Hematol. 8, 224-230; Wong,S., et al. (2001) Oncogene 20, 5644-5659; Phillips J A., Cancer Res.(2000) 52(2):437-43; Harris, A. W., et al, J. Exp. Med. (1988)167(2):353-71; Zeng X X et al., Blood. (1988) 92(10):3529-36; Eriksson,B., et al., Exp. Hematol. (1999) 27(4):682-8; and Kovalchuk, A., et al.,J. Exp. Med. (2000) 192(8):1183-90. Mouse repositories can also be foundat: The Jackson Laboratory, Charles River Laboratories, Taconic, Harlan,Mutant Mouse Regional Resource Centers (MMRRC) National Network and atthe European Mouse Mutant Archive. Such models may be used for in vivotesting of dsRNA, as well as for determining a therapeutically effectivedose.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration. Inpreferred embodiments, the pharmaceutical compositions are administeredby intravenous or intraparenteral infusion or injection.

For oral administration, the dsRNAs useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredients mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredients is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the pharmaceutical compositions of the invention will generally beprovided in sterile aqueous solutions or suspensions, buffered to anappropriate pH and isotonicity. Suitable aqueous vehicles includeRinger's solution and isotonic sodium chloride. In a preferredembodiment, the carrier consists exclusively of an aqueous buffer. Inthis context, “exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of dsRNA inthe cells that express the target gene. Such substances include, forexample, micellar structures, such as liposomes or capsids, as describedbelow. Surprisingly, the present inventors have discovered thatcompositions containing only naked dsRNA and a physiologicallyacceptable solvent are taken up by cells, where the dsRNA effectivelyinhibits expression of the target gene. Although microinjection,lipofection, viruses, viroids, capsids, capsoids, or other auxiliaryagents are required to introduce dsRNA into cell cultures, surprisinglythese methods and agents are not necessary for uptake of dsRNA in vivo.Aqueous suspensions according to the invention may include suspendingagents such as cellulose derivatives, sodium alginate,polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such aslecithin. Suitable preservatives for aqueous suspensions include ethyland n-propyl p-hydroxybenzoate.

The pharmaceutical compositions useful according to the invention alsoinclude encapsulated formulations to protect the dsRNA against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811; PCT publication WO91/06309; and European patent publication EP-A-43075, which areincorporated by reference herein.

In one embodiment, the encapsulated formulation comprises a viral coatprotein. In this embodiment, the dsRNA may be bound to, associated with,or enclosed by at least one viral coat protein. The viral coat proteinmay be derived from or associated with a virus, such as a polyoma virus,or it may be partially or entirely artificial. For example, the coatprotein may be a Virus Protein 1 and/or Virus Protein 2 of the polyomavirus, or a derivative thereof.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions of the invention lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs useful according to the invention can beadministered in combination with other known agents effective intreatment of diseases. In any event, the administering physician canadjust the amount and timing of dsRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

For oral administration, the dsRNAs useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

IV. METHODS FOR TREATING DISEASES CAUSED BY EXPRESSION OF ANANTI-APOPTOTIC GENE

In one embodiment, the invention relates to a method for treating asubject having a disease or at risk of developing a disease caused bythe expression of an anti-apoptotic target gene. In this embodiment, thedsRNA can act as novel therapeutic agents for controlling one or more ofcellular proliferative and/or differentiative disorders. The methodcomprises administering a pharmaceutical composition of the invention tothe patient (e.g., human), such that expression of the target gene issilenced. Because of their high specificity, the dsRNAs of the presentinvention specifically target mRNAs of target genes of diseased cellsand tissues, as described below, and at surprisingly low dosages.

In the prevention of disease, the target gene may be one which isrequired for initiation or maintenance of the disease, or which has beenidentified as being associated with a higher risk of contracting thedisease. In the treatment of disease, the dsRNA can be brought intocontact with the cells or tissue exhibiting the disease. For example,dsRNA comprising a sequence substantially complementary to all or partof an mRNA formed in the transcription of a mutated gene associated withcancer, or one expressed at high levels in tumor cells, e.g. aurorakinase, may be brought into contact with or introduced into a cancerouscell or tumor.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of pancreas, prostate, colon, lung, breast and liverorigin. As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state of condition characterized by rapidlyproliferating cell growth. These terms are meant to include all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. Proliferative disordersalso include hematopoietic neoplastic disorders, including diseasesinvolving hyperplastic/neoplatic cells of hematopoietic origin, e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof.

The present invention also contemplates the simultaneous inhibition ofexpression of other genes. Preferably, other genes are selected whichact additively or synergistically with the inhibition of theanti-apoptotic target gene described above in enhancing the overallaction, for example, in suppressing growth of a cancer cell, or intreating, preventing or managing cancer. Examples of genes which can betargeted for treatment include, without limitation, an oncogene(Hanahan, D. and R. A. Weinberg, Cell (2000) 100:57; and Yokota, J.,Carcinogenesis (2000) 21(3):497-503); genes of proteins that areinvolved in metastasizing and/or invasive processes (Boyd, D., CancerMetastasis Rev. (1996) 15(1):77-89; Yokota, J., Carcinogenesis (2000)21(3):497-503); genes of proteases as well as of molecules that regulateapoptosis and the cell cycle (Matrisian, L. M., Curr. Biol. (1999)9(20):R776-8; Krepela, E., Neoplasma (2001) 48(5):332-49; Basbaum andWerb, Curr. Opin. Cell Biol. (1996) 8:731-738; Birkedal-Hansen, et al.,Crit. Rev. Oral Biol. Med. (1993) 4:197-250; Mignatti and Rifkin,Physiol. Rev. (1993) 73:161-195; Stetler-Stevenson, et al., Annu. Rev.Cell Biol. (1993) 9:541-573; Brinkerhoff, E., and L. M. Matrisan, NatureReviews (2002) 3:207-214; Strasser, A., et al., Annu. Rev. Biochem.(2000) 69:217-45; Chao, D. T. and S. J. Korsmeyer, Annu. Rev. Immunol.(1998) 16:395-419; Mullauer, L., et al., Mutat. Res. (2001)488(3):211-31; Fotedar, R., et al., Prog. Cell Cycle Res. (1996)2:147-63; Reed, J. C., Am. J. Pathol. (2000) 157(5):1415-30; D'Ari, R.,Bioassays (2001) 23(7):563-5); genes that express the EGF receptor;Mendelsohn, J. and J. Baselga, Oncogene (2000) 19(56):6550-65; Normanno,N., et al., Front. Biosci. (2001) 6:D685-707); and the multi-drugresistance 1 gene, MDR1 gene (Childs, S., and V. Ling, Imp. Adv. Oncol.(1994) 21-36).

In one embodiment, a pharmaceutical compositions comprising dsRNA isused to inhibit the expression of the multi-drug resistance 1 gene(“MDR1”). “Multi-drug resistance” (MDR) broadly refers to a pattern ofresistance to a variety of chemotherapeutic drugs with unrelatedchemical structures and different mechanisms of action. Although theetiology of MDR is multifactorial, the overexpression of P-glycoprotein(Pgp), a membrane protein that mediates the transport of MDR drugs,remains the most common alteration underlying MDR in laboratory models(Childs, S., Imp. Adv. Oncol. (1994) 21-36). Moreover, expression of Pgphas been linked to the development of MDR in human cancer, particularlyin the leukemias, lymphomas, multiple myeloma, neuroblastoma, and softtissue sarcoma (Fan., D., et al., Reversal of Multidrug Resistance inCancer, ed. Kellen, J. A. (CRC, Boca Raton, Fla.), pp. 93-125). Recentstudies showed that tumor cells expressing MDR-associated protein (MRP)(Cole, S. P. C., et al., Science (1992) 258:1650-1654) and lungresistance protein (LRP) (Scheffer, G. L., et al., Nat. Med.(1995)1:578-582) and mutation of DNA topoisomerase II (Beck, W. T., J.Natl. Cancer Inst. (1989) 81:1683-1685) also may render MDR.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration. Inpreferred embodiments, the pharmaceutical compositions are administeredby intravenous or intraparenteral infusion or injection.

V. METHODS FOR INHIBITING EXPRESSION OF AN ANTI-APOPTOTIC GENE

In yet another aspect, the invention relates to a method for inhibitingthe expression of an anti-apoptotic gene in an organism. The methodcomprises administering a composition of the invention to the organismsuch that expression of the target anti-apoptotic gene is silenced. Theorganism may be an animal or a plant. Because of their high specificity,the dsRNAs of the present invention specifically target RNAs (primary orprocessed) of target anti-apoptotic genes, and at surprisingly lowdosages. Compositions and methods for inhibiting the expression of thesetarget genes using dsRNAs can be performed as described elsewhereherein.

In one embodiment, the method comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is complementary to at least a part of an RNA transcript of thetarget anti-apoptotic gene of the organism to be treated. When theorganism to be treated is a mammal, such as a human, the composition maybe administered by any means known in the art including, but not limitedto oral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration. Inpreferred embodiments, the compositions are administered by intravenousor intraparenteral infusion or injection.

The methods for inhibiting the expression of a target gene can beapplied to any gene or group of genes that have a direct or indirectinhibitory affect on apoptosis. Examples of human genes which can betargeted for silencing according to the methods of the present inventioninclude, without limitation, an oncogene; a gene that expressesmolecules that induce angiogenesis; genes of proteins that are involvedin metastasizing and/or invasive processes; and genes of proteases aswell as of molecules that regulate apoptosis and the cell cycle. In apreferred embodiment, the tumor disease to be treated is a pancreaticcarcinoma. There is no known treatment for pancreatic cancer, whichcurrently has a survival rate of approximately 3%, the lowest of allcarcinomas.

The methods for inhibition the expression of a target gene can also beapplied to any plant anti-apoptotic gene one wishes to silence, therebyspecifically inhibiting its expression.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3′), an appropriately modified solid support was used forRNA synthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 mL). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl urea. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl esterAB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 mL of toluene, cooled to 0° C. and extracted with three50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated todryness. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated under vacuum and to the residue dichloromethane(50 mL) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Cholesterol Serivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The achieved loading of the CPG was measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) was performed as described inWO 2004/065601, except that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 1.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation^(a) Nucleotide(s) A, a2′-deoxy-adenosine-5′-phosphate, adenosine-5′-phosphate C, c2′-deoxy-cytidine-5′-phosphate, cytidine-5′-phosphate G, g2′-deoxy-guanosine-5′-phosphate, guanosine-5′-phosphate T, t2′-deoxy-thymidine-5′-phosphate, thymidine-5′-phosphate U, u2′-deoxy-uridine-5′-phosphate, uridine-5′-phosphate N, n any2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) am2′-O-methyladenosine-5′-phosphate cm 2′-O-methylcytidine-5′-phosphate gm2′-O-methylguanosine-5′-phosphate tm 2′-O-methyl-thymidine-5′-phosphateum 2′-O-methyluridine-5′-phosphate af2′-fluoro-2′-deoxy-adenosine-5′-phosphate cf2′-fluoro-2′-deoxy-cytidine-5′-phosphate gf2′-fluoro-2′-deoxy-guanosine-5′-phosphate tf2′-fluoro-2′-deoxy-thymidine-5′-phosphate uf2′-fluoro-2′-deoxy-uridine-5′-phosphate A, C, G, T, U, a, underlined:nucleoside-5′-phosphorothioate c, g, t, u am, cm, gm, tm, underlined:2-O-methyl-nucleoside-5′-phosphorothioate um ^(a)capital lettersrepresent 2′-deoxyribonucleotides (DNA), lower case letters representribonucleotides (RNA)

Example 1 Gene Walking of Bcl-2

Selection of Sequences for siRNA Synthesis

Sequential BLAST searches (using the following parameters: Word size 7,Expect value 1000, mismatch penatlty-1) were performed to identifysequences of 23 nucleotides within the sequence of human bcl-2 alpha(GenBank accession number M13994) or bcl-2 beta (GenBank accessionnumber M13995) with 3 or more mismatches to any other human mRNA orgenomic sequence. The 21 nucleotide sequence from position 3 to 23 ofthe 23 mers were used for the synthesis of the sense strands ofapproximately 220 siRNAs. The corresponding antisense strands weresynthesized to comprise a nucleotide sequence fully complementary to the23 mer search query, resulting in a 2-nucleotide single strandedoverhang on the 3′ end of the antisense strand of the siRNA. Thesequences of all siRNAs thus selected and synthesized are shown in Table1, SEQ ID 7 to 444.

The 23 nucleotide sequences thus selected were further compared by BLASTsearching to identify those sequences identically found in mouse bcl-2mRNA, but with 3 or more mismatched to any other mouse mRNA; the searchparameters given above were again used. An siRNA capable of selectivelyinhibiting the expression of bcl-2 both in mice and humans could havecertain advantages in clinical testing.

A dsRNA referred to herein as “K4,” consisting of single strands withthe sequences of SEQ ID 445 and 446, none of which is complementary to asequence of a human mRNA, was synthesized to serve as a null referenceof inhibition. The nucleotide sequence of the sense strand of K4corresponds to nucleotide positions 2608-2630 in the sequence of vectorpEGFP-C1 (GenBank Accession No. U55763).

Creation of Cell Line KB-GFP-BCL2

A reporter cell line for estimating the efficacy of siRNAs in inhibitingthe expression of BCL2 is constructed by transfecting KB cells (ATCCorder number CCL-17) with a reporter construct from which an mRNA istranscribed encoding an eGFP-BCL2 fusion protein. The efficacy ofinhibition may be measured in such cells by comparing the fluorescenceintensity of eGFP in such cells after treatment with an siRNA comprisinga BCL2 sequence with the fluorescence intensity in such cells treatedwith a control siRNA.

The open reading frame of human BCL-2 (alpha splice form, GenBankaccession number M13994) is PCR-amplified from a human BD™Marathon-Ready cDNA library (BD Biosciences Clontech, Palo Alto, Calif.,USA, Cat. #: 639343) using the BD Advantage HF 2 PCR kit (BD BiosciencesClontech, Palo Alto, Calif., USA Cat. #: 639123). Primer, nucleotide andenzyme concentration are used according to manufacturer's instructions.Amplification is performed in 30 cycles with the following three stepsin each cycle: 20 sec. 95° C., 30 sec. 62° C., 60 sec. 72° C. A finalstep of 120 sec at 72° C. terminates the amplification reaction. Primersare AAA CTC GAG GCG CAC GCT GGG AGA ACG GGG (SEQ ID NO:490) (introducinga XhoI (italics) restriction site upstream of the codon coding for aminoacid 2 of BCL2) and AAA TCT AGA TCA CTT GTG GCT CAG ATA GGC (SEQ IDNO:491) (introducing a XbaI restriction site (italics) after the BCL2stop codon (double underlined)). The PCR product is gel-purified on a0.8% agarose gel, digested with XhoI and XbaI and ligated into pEGFP-C3(BD Biosciences Clontech, Palo Alto Calif., USA; Cat. #: 632315)digested with XhoI and XbaI. The correct insertion of the cDNA isverified by sequencing. The plasmid is transfected into KB cells (ATCCorder no. CCL17) by lipofection with Lipofectamin 2000 andNeomycin-resistant and fluorescing clones are identified in three roundsof: selection in the presence of G418 for 48 h followed by selection offluorescent cells and replating of single cells using FACS-analysis.

Introduction of siRNAs into KB-GFP-BCL2-Cells and Determination of GeneExpression Inhibition by FACS Analysis

KB-GFP-BCL2-Cells (about 80% confluent) were trypsinized from 96 mmPetri dishes with 5 ml trypsin-EDTA (0.25% Trypsin; 1 mM Na₄-EDTA;Gibco/Invitrogen, Karlsruhe, Germany) for 3-5 min at 37° C. The trypsinsolution was gently removed, 5 ml cell culture medium (RPMI 1640supplemented with 10% FCS, both Biochrom A G Berlin, Germany) were addedand cells were centrifuged at 400 g for 5 minutes at room temperature.The cell pellet was resuspended in 250 μl cell culture medium and thecell number per unit volume determined in a Neubauer chamber. Theresuspended cells were diluted to a density of 4 millions of cells perml cell culture medium and 500 μl of this suspension were added to a 0.4cm cuvette (Gene Pulser Cuvette, Bio-Rad Laboratories, Inc., Hercules,USA). 5 μl of a 20 μM stock solution of the respective siRNA inannealing buffer (100 mM NaCl, 10 mM Na₂HPO₄, 10 mM NaH₂PO₄, pH 6.8) wasadded to the cell suspension, for a total concentration of the siRNA inthe incubation mixture of 200 nM, and gentle mixing was achieved byrepeated aspiration/expulsion of the suspension using a 1 ml pipette(100-1000, Eppendorf A G, Hamburg, Germany). Electroporation wasperformed at 250V and 2500 μF with an exponential pulse in a Gene PulserX cell with CE module (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA). 200 μl of the suspension were seeded in one well of a6-well-plate, 2 ml cell culture medium per well were added and theplates were incubated at 37° C. and 5% CO₂ for 48 h (Heracell incubator,Kendro Laboratory products GmbH, Langenselbold, Germany).

Cells were harvested by removing the cell culture medium, adding 500 μltrypsin-EDTA (Gibco-Invitrogen GmbH, Karlsruhe, Germany) per well andincubating for 3-5 min at 37° C. After removing the trypsin solution andresuspending cells in 500 μl cell culture medium, the suspension wastransferred to FACS tubes (5 ml, Sarstedt A G & Co., Nümbrecht, Germany)and centrifuged at 400 g for 5 min. Pellets were resuspended in 1 ml PBS(Biochrom, Cambridge, UK) and eGFP-fluorescence was measured byflow-cytometry (XL-MCL, Beckman Coulter GmbH, Krefeld, Germany) 10.000cells were counted per tube and the percentage of eGFP-positive cellswas multiplied with the mean fluorescence intensity of all measuredcells to yield an overall fluorescence intensity per 10.000 cells(FI₁₀₀₀₀).

Inhibition of expression of the eGFP-BCL2 fusion protein by the varioussiRNA species is summarized in Table 2. Therein, the efficacy ininhibiting the expression of eGFP-BCL2 is expressed as the amount bywhich FI₁₀₀₀₀ is reduced by incubation of KB-GFP-BCL2-cells withindividual siRNAs compared to incubation with the unrelated referencesiRNA K4, as given by the following equation

${Efficacy} = {\left( {1 - \frac{{FI}_{10000}({siRNA})}{{FI}_{10000}\left( {K\; 4} \right)}} \right) \times 100\%}$

Triplicate determinations were performed for each siRNA species toobtain average values and standard deviations.

The average transfection efficiency of the above method was estimatedseparately by transfecting unmodified KB cells (ATCC order numberCCL-17) with plasmid pEGFP-C1, using 1 μg plasmid in 2 μl TE buffer (100mM Tris-HCl, pH 7.3, 10 mM EDTA, pH 8.0) in place of the siRNA stocksolution in the above procedure. Average transfection efficiency wasestimated as 80±5%.

TABLE 2 List of siRNAs employed in the identification of siRNAs capableof efficiently inhi- biting the expression of bcl2 in mammalian cells.Columns refer to: the denomination given to the siRNA, the number of thenucleotide within the human bcl-2 mRNA se- quence, counting from its5′-end, which marks the start of the 23mer sequence which the antisensestrand of the siRNA is complementary to, the sequences of the sense (s)and the antisense (as) strand of the siRNA, the specificity for humanbcl-2, human and mouse bcl-2 or the human splice variant bcl-2α, theefficacy of gene ex- pression inhibition of human bcl-2 as determined bythe FACS assay described herein- above, given as % inhibition,± standard deviation, in comparison to cells trans- fected with the nullcontrol siRNA denominated K4 and derived from the mRNA of the neomycinresistance gene, and the SEQ. ID of the strand sequences 5′-Start Namenucleotide Sequence Specificity Efficacy Seq. ID B31 31 s:5′-ccgggagauagugaugaagua-3′ human  84 ± 1 SEQ ID NO: 7 as:3′-uuggcccucuaucacuacuucau-5′ SEQ ID NO: 8 B529 529 s:5′-gacugaguaccugaaccggca-3′ human +  81 ± 0 SEQ ID NO: 9 as:3′-uacugacucauggacuuggccgu-5′ mouse SEQ ID NO: 10 B25 25 s:5′-cgauaaccgggagauagugau-3′ human  80 ± 1 SEQ ID NO: 11 as:3′-augcuauuggcccucuaucacua-5′ SEQ ID NO: 12 B21 21 s:5′-gguacgauaaccgggagauag-3′ human  79 ± 2 SEQ ID NO: 13 as:3′-ccccaugcuauuggcccucuauc-5′ SEQ ID NO: 14 B22 22 s:5′-guacgauaaccgggagauagu-3′ human  79 ± 1 SEQ ID NO: 15 as:3′-cccaugcuauuggcccucuauca-5′ SEQ ID NO: 16 B522 522 s:5′-uguggaugacugaguaccuga-3′ human  79 ± 3 SEQ ID NO: 17 as:3′-ggacaccuacugacucauggacu-5′ SEQ ID NO: 18 B463 463 s:5′-ggucauguguguggagagcgu-3′ human  78 ± 0 SEQ ID NO: 19 as:3′-ccccaguacacacaccucucgca-5′ SEQ ID NO: 20 B523 523 s:5′-guggaugacugaguaccugaa-3′ human +  75 ± 2 SEQ ID NO: 21 as:3′-gacaccuacugacucauggacuu-5′ mouse SEQ ID NO: 22 B519 519 s:5′-cccuguggaugacugaguacc-3′ human +  73 ± 0 SEQ ID NO: 23 as:3′-gcgggacaccuacugacucaugg-5′ mouse SEQ ID NO: 24 B522 522 s:5′-uguggaugacugaguaccuga-3′ human  71 ± 3 SEQ ID NO: 25 as:3′-ggacaccuacugacucauggacu-5′ SEQ ID NO: 26 B133 133 s:5′-accgggcaucuucuccuccca-3′ human  70 ± 1 SEQ ID NO: 27 as:3′-cguggcccguagaagaggagggu-5′ SEQ ID NO: 28 B442 442 s:5′-ggccuucuuugaguucggugg-3′ human +  70 ± 5 SEQ ID NO: 29 as:3′-caccggaagaaacucaagccacc-5′ mouse SEQ ID NO: 30 B531 531 s:5′-cugaguaccugaaccggcacc-3′ human  70 ± 3 SEQ ID NO: 31 as:3′-cugacucauggacuuggccgugg-5′ SEQ ID NO: 32 B440 440 s:5′-guggccuucuuugaguucggu-3′ human +  69 ± 1 SEQ ID NO: 33 as:3′-aacaccggaagaaacucaagcca-5′ mouse SEQ ID NO: 34 B54 54 s:5′-uccauuauaagcugucgcaga-3′ human  69 ± 1 SEQ ID NO: 35 as:3′-guagguaauauucgacagcgucu-5′ SEQ ID NO: 36 B461 461 s:5′-ggggucauguguguggagagc-3′ human  69 ± 1 SEQ ID NO: 37 as:3′-caccccaguacacacaccucucg-5′ SEQ ID NO: 38 B525 525 s:5′-ggaugacugaguaccugaacc-3′ human +  68 ± 8 SEQ ID NO: 39 as:3′-caccuacugacucauggacuugg-5′ mouse SEQ ID NO: 40 B535 535 s:5′-guaccugaaccggcaccugca-3′ human  68 ± 8 SEQ ID NO: 41 as:3′-cucauggacuuggccguggacgu-5′ SEQ ID NO: 42 B508 508 s:5′-ggacaacaucgcccuguggau-3′ human +  67 ± 2 SEQ ID NO: 43 as:3′-caccuguuguagcgggacaccua-5′ mouse SEQ ID NO: 44 B56 56 s:5′-cauuauaagcugucgcagagg-3′ human  67 ± 1 SEQ ID NO: 45 as:3′-agguaauauucgacagcgucucc-5′ SEQ ID NO: 46 B462 462 s:5′-gggucauguguguggagagcg-3′ human +  66 ± 3 SEQ ID NO: 47 as:3′-accccaguacacacaccucucgc-5′ mouse SEQ ID NO: 48 B33 33 s:5′-gggagauagugaugaaguaca-3′ human  66 ± 6 SEQ ID NO: 49 as:3′-ggcccucuaucacuacuucaugu-5′ SEQ ID NO: 50 B466 466 s:5′-cauguguguggagagcgucaa-3′ human +  64 ± 1 SEQ ID NO: 51 as:3′-caguacacacaccucucgcaguu-5′ mouse SEQ ID NO: 52 B459 459 s:5′-guggggucauguguguggaga-3′ human  63 ± 5 SEQ ID NO: 53 as:3′-gccaccccaguacacacaccucu-5′ SEQ ID NO: 54 B45 45 s:5′-ugaaguacauccauuauaagc-3′ human  63 ± 3 SEQ ID NO: 55 as:3′-cuacuucauguagguaauauucg-5′ SEQ ID NO: 56 B520 520 s:5′-ccuguggaugacugaguaccu-3′ human +  62 ± 2 SEQ ID NO: 57 as:3′-cgggacaccuacugacucaugga-5′ mouse SEQ ID NO: 58 B465 465 s:5′-ucauguguguggagagcguca-3′ human +  61 ± 7 SEQ ID NO: 59 as:3′-ccaguacacacaccucucggagu-5′ mouse SEQ ID NO: 60 B517 517 s:5′-cgcccuguggaugacugagua-3′ human +  61 ± 3 SEQ ID NO: 61 as:3′-uagcgggacaccuacugacucau-5′ mouse SEQ ID NO: 62 B524 524 s:5′-uggaugacugaguaccugaac-3′ human +  61 ± 2 SEQ ID NO: 63 as:3′-acaccuacugacucauggacuug-5′ mouse SEQ ID NO: 64 B555 555 s:5′-acaccuggauccaggauaacg-3′ human +  60 ± 4 SEQ ID NO: 65 as:3′-cguguggaccuagguccuauugc-5′ mouse SEQ ID NO: 66 B583 583 s:5′-ggaugccuuuguggaacugua-3′ human  60 ± 5 SEQ ID NO: 67 as:3′-acccuacggaaacaccuugacau-5′ SEQ ID NO: 68 B464 464 s:5′-gucauguguguggagagcguc-3′ human +  59 ± 4 SEQ ID NO: 69 as:3′-cccaguacacacaccucucgcag-5′ mouse SEQ ID NO: 70 B619 619 s:5′-gccucuguuugauuucuccug-3′ human α  59 ± 4 SEQ ID NO: 71 as:3′-gccggagacaaacuaaagaggac-5′ SEQ ID NO: 72 B617 617 s:5′-cggccucuguuugauuucucc-3′ humanα  59 ± 1 SEQ ID NO: 73 as:3′-acgccggagacaaacuaaagagg-5′ SEQ ID NO: 74 B77 77 s:5′-ggcuacgagugggaugcggga-3′ human  59 ± 6 SEQ ID NO: 75 as:3′-ccccgaugcucacccuacgcccu-5′ SEQ ID NO: 76 B19 19 s:5′-aggguacgauaaccgggagau-3′ human  58 ± 3 SEQ ID NO: 77 as:3′-ugucccaugcuauuggcccucua-5′ SEQ ID NO: 78 B18 18 s:5′-caggguacgauaaccgggaga-3′ human  57 ± 8 SEQ ID NO: 79 as:3′-uugucccaugcuauuggcccucu-5′ SEQ ID NO: 80 B457 457 s:5′-cgguggggucaugugugugga-3′ human +  57 ± 3 SEQ ID NO: 81 as:3′-aagccaccccaguacacacaccu-5′ mouse SEQ ID NO: 82 B24 24 s:5′-acgauaaccgggagauaguga-3′ human  56 ± 1 SEQ ID NO: 83 as:3′-caugcuauuggcccucuaucacu-5′ SEQ ID NO: 84 B411 411 s:5′-uggccuucuuugaguucggug-3′ human +  56 ± 4 SEQ ID NO: 85 as:3′-acaccggaagaaacucaagccac-5′ mouse SEQ ID NO: 86 B32 32 s:5′-cgggagauagugaugaaguac-3′ human  56 ± 4 SEQ ID NO: 87 as:3′-uggcccucuaucacuacuucaug-5′ SEQ ID NO: 88 B47 47 s:5′-aaguacauccauuauaagcug-3′ human  56 ± 1 SEQ ID NO: 89 as:3′-acuucauguagguaauauucgac-5′ SEQ ID NO: 90 B52 52 s:5′-cauccauuauaagcugucgca-3′ human  56 ± 3 SEQ ID NO: 91 as:3′-auguagguaauauucgacagcgu-5′ SEQ ID NO: 92 B439 439 s:5′-uguggccuucuuugaguucgg-3′ human +  55 ± 7 SEQ ID NO: 93 as:3′-uaacaccggaagaaacucaagcc-5′ mouse SEQ ID NO: 94 B79 79 s:5′-cuacgagugggaugcgggaga-3′ human  55 ± 9 SEQ ID NO: 95 as:3′-ccgaugcucacccuacgcccucu-5′ SEQ ID NO: 96 B44 44 s:5′-augaaguacauccauuauaag-3′ human  55 ± 5 SEQ ID NO: 97 as:3′-acuacuucauguagguaauauuc-5′ SEQ ID NO: 98 B443 443 s:5′-gccuucuuugaguucgguggg-3′ human +  54 ± 4 SEQ ID NO: 99 as:3′-accggaagaaacucaagccaccc-5′ mouse SEQ ID NO: 100 B467 467 s:5′-auguguguggagagcgucaac-3′ human +  54 ± 3 SEQ ID NO: 101 as:3′-aguacacacaccucucgcaguug-5′ mouse SEQ ID NO: 102 B28 28 s:5′-uaaccgggagauagugaugaa-3′ human  54 ± 3 SEQ ID NO: 103 as:3′-cuauuggcccucuaucacuacuu-5′ SEQ ID NO: 104 B521 521 s:5′-cuguggaugacugaguaccug-3′ human  54 ± 1 SEQ ID NO: 105 as:3′-gggacaccuacugacucauggac-5′ SEQ ID NO: 106 B302 302 s:5′-gacgacuucucccgccgcuac-3′ human  54 ± 1 SEQ ID NO: 107 as:3′-cgcugcugaagagggcggcgaug-5′ SEQ ID NO: 108 B444 444 s:5′-ccuucuuugaguucggugggg-3′ human +  53 ± 2 SEQ ID NO: 109 as:3′-ccggaagaaacucaagccacccc-5′ mouse SEQ ID NO: 110 B509 509 s:5′-gacaacaucgcccuguggaug-3′ human +  53 ± 5 SEQ ID NO: 111 as:3′-accuguuguagcgggacaccuac-5′ mouse SEQ ID NO: 112 B468 468 s:5′-uguguguggagagcgucaacc-3′ human  53 ± 1 SEQ ID NO: 113 as:3′-guacacacaccucucgcaguugg-5′ SEQ ID NO: 114 B518 518 s:5′-gcccuguggaugacugaguac-3′ human +  52 ± 4 SEQ ID NO: 115 as:3′-agcgggacaccuacugacucaug-5′ mouse SEQ ID NO: 116 B55 55 s:5′-ccauuauaagcugucgcagag-3′ human  52 ± 3 SEQ ID NO: 117 as:3′-uagguaauauucgacagcgucuc-5′ SEQ ID NO: 118 B586 586 s:5′-ugccuuuguggaacuguacgg-3′ human  52 ± 9 SEQ ID NO: 119 as:3′-cuacggaaacaccuugacaugcc-5′ SEQ ID NO: 120 B445 445 s:5′-cuucuuugaguucgguggggu-3′ human +  51 ± 3 SEQ ID NO: 121 as:3′-cggaagaaacucaagccacccca-5′ mouse SEQ ID NO: 122 B526 526 s:5′-gaugacugaguaccugaaccg-3′ human +  51 ± 1 SEQ ID NO: 123 as:3′-accuacugacucauggacuuggc-5′ mouse SEQ ID NO: 124 B328 328 s:5′-cgacuucgccgagauguccag-3′ human  51 ± 1 SEQ ID NO: 125 as:3′-ccgcugaagcggcucuacagguc-5′ SEQ ID NO: 126 B327 327 s:5′-gcgacuucgccgagaugucca-3′ human  51 ± 4 SEQ ID NO: 127 as:3′-ggcgcugaagcggcucuacaggu-5′ SEQ ID NO: 128 B460 460 s:5′-uggggucauguguguggagag-3′ human  51 ± 2 SEQ ID NO: 129 as:3′-ccaccccaguacacacaccucuc-5′ SEQ ID NO: 130 B302 302 s:5′-gacgacuucucccgccgcuac-3′ human  49 ± 1 SEQ ID NO: 131 as:3′-cgcugcugaagagggcggcgaug-5′ SEQ ID NO: 132 B30 30 s:5′-accgggagauagugaugaagu-3′ human  49 ± 1 SEQ ID NO: 133 as:3′-auuggcccucuaucacuacuuca-5′ SEQ ID NO: 134 B30 30 s:5′-accgggagauagugaugaagu-3′ human  49 ± 1 SEQ ID NO: 135 as:3′-auuggcccucuaucacuacuuca-5′ SEQ ID NO: 136 B5 5 s:5′-cacgcugggagaacgggguac-3′ human  48 ± 1 SEQ ID NO: 137 as:3′-gcgugcgacccucuugccccaug-5′ SEQ ID NO: 138 B76 76 s:5′-gggcuacgagugggaugcggg-3′ human  48 ± 2 SEQ ID NO: 139 as:3′-uccccgaugcucacccuacgccc-5′ SEQ ID NO: 140 B514 514 s:5′-caucgcccuguggaugacuga-3′ human +  46 ± 2 SEQ ID NO: 141 as:3′-uuguagcgggacaccuacugacu-5′ mouse SEQ ID NO: 142 B510 510 s:5′-acaacaucgcccuguggauga-3′ human +  45 ± 1 SEQ ID NO: 143 as:3′-ccuguuguagcgggacaccuacu-5′ mouse SEQ ID NO: 144 B301 301 s:5′-cgacgacuucucccgccgcua-3′ human  45 ± 2 SEQ ID NO: 145 as:3′-ccgcugcugaagagggcggcgau-5′ SEQ ID NO: 146 B11 11 s:5′-gggagaacgggguacgacaac-3′ human  45 ± 2 SEQ ID NO: 147 as:3′-gacccucuugccccaugcuguug-5′ SEQ ID NO: 148 B472 472 s:5′-uguggagagcgucaaccggga-3′ human  45 ± 11 SEQ ID NO: 149 as:3′-acacaccucucgcaguuggcccu-5′ SEQ ID NO: 150 B475 475 s:5′-ggagagcgucaaccgggagau-3′ human  44 ± 1 SEQ ID NO: 151 as:3′-caccucucgcaguuggcccucua-5′ SEQ ID NO: 152 B469 469 s:5′-guguguggagagcgucaaccg-3′ human  44 ± 2 SEQ ID NO: 153 as:3′-uacacacaccucucgcaguuggc-5′ SEQ ID NO: 154 B135 135 s:5′-cgggcaucuucuccucccagc-3′ human  42 ± 4 SEQ ID NO: 155 as:3′-uggcccguagaagaggagggucg-5′ SEQ ID NO: 156 B559 559 s:5′-cuggauccaggauaacggagg-3′ human +  42 ± 2 SEQ ID NO: 157 as:3′-uggaccuagguccuauugccucc-5′ mouse SEQ ID NO: 158 B46 46 s:5′-gaaguacauccauuauaagcu-3′ human  42 ± 2 SEQ ID NO: 159 as:3′-uacuucauguagguaauauucga-5′ SEQ ID NO: 160 B616 616 s:5′-gcggccucuguuugauuucuc-3′ humanα  42 ± 4 SEQ ID NO: 161 as:3′-uacgccggagacaaacuaaagag-5′ SEQ ID NO: 162 B332 332 s:5′-uucgccgagauguccagccag-3′ human  42 ± 3 SEQ ID NO: 163 as:3′-ugaagcggcucuacaggucgguc-5′ SEQ ID NO: 164 B53 53 s:5′-auccauuauaagcugucgcag-3′ human  42 ± 1 SEQ ID NO: 165 as:3′-uguagguaauauucgacagcguc-5′ SEQ ID NO: 166 B474 474 s:5′-uggagagcgucaaccgggaga-3′ human  40 ± 9 SEQ ID NO: 167 as:3′-acaccucucgcaguuggcccucu-5′ SEQ ID NO: 168 B654 654 s:5′-ggagagcgucaaccgggagau-3′ human  40 ± 2 SEQ ID NO: 169 as:3′-caccucucgcaguuggcccucua-5′ SEQ ID NO: 170 B470 470 s:5′-uguguggagagcgucaaccgg-3′ human  39 ± 3 SEQ ID NO: 171 as:3′-acacacaccucucgcaguuggcc-5′ SEQ ID NO: 172 B330 330 s:5′-acuucgccgagauguccagcc-3′ human  38 ± 3 SEQ ID NO: 173 as:3′-gcugaagcggcucuacaggucgg-5′ SEQ ID NO: 174 B29 29 s:5′-aaccgggagauagugaugaag-3′ human  38 ± 2 SEQ ID NO: 175 as:3′-uauuggcccucuaucacuacuuc-5′ SEQ ID NO: 176 B668 668 s:5′-gcccuggugggagcuugcauc-3′ human α  37 ± 3 SEQ ID NO: 179 as:3′-accgggaccacccucgaacguag-5′ SEQ ID NO: 180 B668 668 s:5′-gcccuggugggagcuugcauc-3′ human α  37 ± 3 SEQ ID NO: 177 as:3′-accgggaccacccucgaacguag-5′ SEQ ID NO: 178 B507 507 s:5′-uggacaacaucgcccugugga-3′ human +  36 ± 4 SEQ ID NO: 181 as:3′-ccaccuguuguagcgggacaccu-5′ mouse SEQ ID NO: 182 B511 511 s:5′-caacaucgcccuguggaugac-3′ human +  35 ± 1 SEQ ID NO: 183 as:3′-cuguuguagcgggacaccuacug-5′ mouse SEQ ID NO: 184 B7 7 s:5′-cgcugggagaacgggguacga-3′ human  35 ± 5 SEQ ID NO: 185 as:3′-gugcgacccucuugccccaugcu-5′ SEQ ID NO: 186 B556 556 s:5′-caccuggauccaggauaacgg-3′ human +  35 ± 2 SEQ ID NO: 187 as:3′-guguggaccuagguccuauugcc-5′ mouse SEQ ID NO: 188 B516 516 s:5′-ucgcccuguggaugacugagu-3′ human +  34 ± 2 SEQ ID NO: 189 as:3′-guagcgggacaccuacugacuca-5′ mouse SEQ ID NO: 190 B557 557 s:5′-accuggauccaggauaacgga-3′ human +  34 ± 1 SEQ ID NO: 191 as:3′-uguggaccuagguccuauugccu-5′ mouse SEQ ID NO: 192 B321 321 s:5′-accgccgcgacuucgccgaga-3′ human  34 ± 3 SEQ ID NO: 193 as:3′-gauggcggcgcugaagcggcucu-5′ SEQ ID NO: 194 B447 447 s:5′-ucuuugaguucggugggguca-3′ human +  32 ± 3 SEQ ID NO: 195 as:3′-gaagaaacucaagccaccccagu-5′ mouse SEQ ID NO: 196 B515 515 s:5′-aucgcccuguggaugacugag-3′ human +  32 ± 3 SEQ ID NO: 197 as:3′-uguagcgggacaccuacugacuc-5′ mouse SEQ ID NO: 198 B558 558 s:5′-ccuggauccaggauaacggag-3′ human +  32 ± 1 SEQ ID NO: 199 as:3′-guggaccuagguccuauugccuc-5′ mouse SEQ ID NO: 200 B446 446 s:5′-uucuuugaguucggugggguc-3′ human +  31 ± 2 SEQ ID NO: 201 as:3′-ggaagaaacucaagccaccccag-5′ mouse SEQ ID NO: 202 B527 527 s:5′-augacugaguaccugaaccgg-3′ human +  31 ± 2 SEQ ID NO: 203 as:3′-ccuacugacucauggacuuggcc-5′ mouse SEQ ID NO: 204 B381 381 s:5′-gacgcuuugccacgguggugg-3′ human +  30 ± 3 SEQ ID NO: 205 as:3′-cccugcgaaacggugccaccacc-5′ mouse SEQ ID NO: 206 B27 27 s:5′-auaaccgggagauagugauga-3′ human  30 ± 4 SEQ ID NO: 207 as:3′-gcuauuggcccucuaucacuacu-5′ SEQ ID NO: 208 B530 530 s:5′-acugaguaccugaaccggcac-3′ human  30 ± 7 SEQ ID NO: 209 as:3′-acugacugauggacuuggccgug-5′ SEQ ID NO: 210 B10 10 s:5′-ugggagaacaggguacgacaa-3′ human  29 ± 1 SEQ ID NO: 211 as:3′-cgacccucuugucccaugcuguu-5′ SEQ ID NO: 212 B132 132 s:5′-caccgggcaucuucuccuccc-3′ human  29 ± 3 SEQ ID NO: 213 as:3′-gcguggcccguagaagaggaggg-5′ SEQ ID NO: 214 B380 380 s:5′-ggacgcuuugccacgguggug-3′ human +  29 ± 3 SEQ ID NO: 215 as:3′-ccccugcgaaacggugccaccac-5′ mouse SEQ ID NO: 216 B452 452 s:5′-gaguucgguggggucaugugu-3′ human +  29 ± 7 SEQ ID NO: 217 as:3′-aacucaagccaccccaguacaca-5′ mouse SEQ ID NO: 218 B383 383 s:5′-cgcuuugccacggugguggag-3′ human +  29 ± 0 SEQ ID NO: 219 as:3′cugcgaaacggugccaccaccac5′ mouse SEQ ID NO: 220 B51 51 s:5′-acauccauuauaagcugucgc-3′ human  29 ± 4 SEQ ID NO: 221 as:3′-cauguagguaauauucgacagcg-5′ SEQ ID NO: 222 B82 82 s:5′-cgagugggaugcgggagaugu-3′ human  29 ± 3 SEQ ID NO: 223 as:3′-augcucacccuacgcccucuaca-5′ SEQ ID NO: 224 B380 380 s:5′-ggacgcuuugccacgguggug-3′ human +  29 ± 3 SEQ ID NO: 225 as:3′-ccccugcgaaacggugccaccac-5′ mouse SEQ ID NO: 226 B51 51 s:5′-acauccauuauaagcugucgc-3′ human  29 ± 4 SEQ ID NO: 227 as:3′-cauguagguaauauucgacagcg-5′ SEQ ID NO: 228 B513 513 s:5′-acaucgcccuguggaugacug-3′ human +  28 ± 1 SEQ ID NO: 229 as:3′-guuguagcgggacaccuacugac-5′ mouse SEQ ID NO: 230 B49 49 s:5′-guacauccauuauaagcuguc-3′ human  28 ± 5 SEQ ID NO: 231 as:3′-uucauguagguaauauucgacag-5′ SEQ ID NO: 232 B554 554 s:5′-cacaccuggauccaggauaac-3′ human +  27 ± 3 SEQ ID NO: 233 as:3′-acguguggaccuagguccuauug-5′ mouse SEQ ID NO: 234 B326 326 s:5′-cgcgacuucgccgagaugucc-3′ human  27 ± 4 SEQ ID NO: 235 as:3′-cggcgcugaagcggcucuacagg-5′ SEQ ID NO: 236 B528 528 s:5′-ugacugaguaccugaaccggc-3′ human +  26 ± 2 SEQ ID NO: 237 as:3′-cuacugacucauggacuuggccg-5′ mouse SEQ ID NO: 238 B560 560 s:5′-uggauccaggauaacggaggc-3′ human +  26 ± 5 SEQ ID NO: 239 as:3′-ggaccuagguccuauugccuccg-5′ mouse SEQ ID NO: 240 B78 78 s:5′-gcuacgagugggaugcgggag-3′ human  26 ± 4 SEQ ID NO: 241 as:3′-cccgaugcucacccuacgcccuc-5′ SEQ ID NO: 242 B451 451 s:5′-ugaguucgguggggucaugug-3′ human +  25 ± 12 SEQ ID NO :243 as:3′-aaacucaagccaccccaguacac-5′ mouse SEQ ID NO: 244 B454 454 s:5′-guucgguggggucaugugugu-3′ human +  25 ± 5 SEQ ID NO: 245 as:3′-cucaagccaccccaguacacaca-5′ mouse SEQ ID NO: 246 B569 569 s:5′-gauaacggaggcugggaugcc-3′ humanα +  25 ± 2 SEQ ID NO: 247 as:3′-uccuauugccuccgacccuacgg-5′ mouse SEQ ID NO: 248 B670 670 s:5′-ccuggugggagcuugcaucac-3′ human α  25 ± 4 SEQ ID NO: 249 as:3′-cgggaccacccucgaacguagug-5′ SEQ ID NO: 250 B75 75 s:5′-ggggcuacgagugggaugcgg-3′ human  25 ± 5 SEQ ID NO: 251 as:3′-cuccccgaugcucacccuacgcc-5′ SEQ ID NO: 252 B23 23 s:5′-uacgacaaccgggagauagug-3′ human  24 ± 1 SEQ ID NO: 253 as:3′-ccaugcuguuggcccucuaucac-5′ SEQ ID NO: 254 B295 295 s:5′-ggccggcgacgacuucucccg-3′ human  24 ± 7 SEQ ID NO: 255 as:3′-guccggccgcugcugaagagggc-5′ SEQ ID NO: 256 B329 329 s:5′-gacuucgccgagauguccagc-3′ human  24 ± 2 SEQ ID NO: 257 as:3′-cgcugaagcggcucuacaggucg-5′ SEQ ID NO: 258 B505 505 s:5′-gguggacaacaucgcccugug-3′ human +  24 ± 3 SEQ ID NO: 259 as:3′-gaccaccuguuguagcgggacac-5′ mouse SEQ ID NO: 260 B81 81 s:5′-acgagugggaugcgggagaug-3′ human  24 ± 2 SEQ ID NO: 261 as:3′-gaugcucacccuacgcccucuac-5′ SEQ ID NO: 262 B134 134 s:5′-ccgggcaucuucuccucccag-3′ human  23 ± 6 SEQ ID NO: 263 as:3′-guggcccguagaagaggaggguc-5′ SEQ ID NO: 264 B540 540 s:5′-ugaaccggcaccugcacaccu-3′ human  23 ± 2 SEQ ID NO: 265 as:3′-ggacuuggccguggacgugugga-5′ SEQ ID NO: 266 B458 458 s:5′-gguggggucauguguguggag-3′ human  23 ± 6 SEQ ID NO: 267 as:3′-agccaccccaguacacacaccuc-5′ SEQ ID NO: 268 B448 448 s:5′-cuuugaguucgguggggucau-3′ human +  22 ± 9 SEQ ID NO: 269 as:3′-aagaaacucaagccaccccagua-5′ mouse SEQ ID NO: 270 B671 671 s:5′-cuggugggagcuugcaucacc-3′ human α  22 ± 3 SEQ ID NO: 271 as:3′-gggaccacccucgaacguagugg-5′ SEQ ID NO: 272 B323 323 s:5′-cgccgcgacuucgccgagaug-3′ human  22 ± 2 SEQ ID NO: 273 as:3′-uggcggcgcugaagcggcucuac-5′ SEQ ID NO: 274 B4 4 s:5′-gcacgcugggagaacggggua-3′ human  21 ± 1 SEQ ID NO: 275 as:3′-cgcgugcgacccucuugccccau-5′ SEQ ID NO: 276 B453 453 s:5′-aguucgguggggucaugugug-3′ human +  21 ± 11 SEQ ID NO: 277 as:3′-acucaagccaccccaguacacac-5′ mouse SEQ ID NO: 278 B6 6 s:5′-acgcugggagaacgggguacg-3′ human  21 ± 4 SEQ ID NO: 279 as:3′-cgugcgacccucuugccccaugc-5′ SEQ ID NO: 280 B659 659 s:5′-cucaguuuggcccugguggga-3′ human α  21 ± 4 SEQ ID NO: 281 as:3′-gcgagucaaaccgggaccacccu-5′ SEQ ID NO: 282 B50 50 s:5′-uacauccauuauaagcugucg-3′ human  21 ± 0 SEQ ID NO: 283 as:3′-ucauguagguaauauucgacagc-5′ SEQ ID NO: 284 B334 334 s:5′-cgccgagauguccagccagcu-3′ human  21 ± 5 SEQ ID NO: 285 as:3′-aagcggcucuacaggucggucga-5′ SEQ ID NO: 286 B659 659 s:5′-cucaguuuggcccugguggga-3′ human α  21 ± 4 SEQ ID NO: 287 as:3′-gcgagucaaaccgggaccacccu-5′ SEQ ID NO: 288 B289 289 s:5′-ccgccaggccggcgacgacuu-3′ human  20 ± 3 SEQ ID NO: 289 as:3′-gaggcgguccggccgcugcugaa-5′ SEQ ID NO: 290 B384 384 s:5′-gcuuugccacggugguggagg-3′ human +  20 ± 2 SEQ ID NO: 291 as:3′-ugcgaaacggugccaccaccucc-5′ mouse SEQ ID NO: 292 B48 48 s:5′-aguacauccauuauaagcugu-3′ human  20 ± 3 SEQ ID NO: 293 as:3′-cuucauguagguaauauucgaca-5′ SEQ ID NO: 294 B538 538 s:5′-ccugaaccggcaccugcacac-3′ human  20 ± 4 SEQ ID NO: 295 as:3′-auggacuuggccguggacgugug-5′ SEQ ID NO: 296 B324 324 s:5′-gccgcgacuucgccgagaugu-3′ human  19 ± 6 SEQ ID NO: 297 as:3′-ggcggcgcugaagcggcucuaca-5′ SEQ ID NO: 298 B12 12 s:5′-ggagaacgggguacgacaacc-3′ human  19 ± 3 SEQ ID NO: 299 as:3′-acccucuugccccaugcuguugg-5′ SEQ ID NO: 300 B13 13 s:5′-gagaacgggguacgacaaccg-3′ Human  18 ± 3 SEQ ID NO: 301 as:3′-cccucuugccccaugcuguuggc-5′ SEQ ID NO: 302 B352 352 s:5′-gcugcaccugacgcccuucac-3′ human +  18 ± 5 SEQ ID NO: 303 as:3′-gucgacguggacugcgggaagug-5′ mouse SEQ ID NO: 304 B676 676 s:5′-gggagcuugcaucacccuggg-3′ humanα  18 ± 3 SEQ ID NO: 305 as:3′-cacccucgaacguagugggaccc-5′ SEQ ID NO: 306 B325 325 s:5′-ccgcgacuucgccgagauguc-3′ human  18 ± 1 SEQ ID NO: 307 as:3′-gcggcgcugaagcggcucuacag-5′ SEQ ID NO: 308 B322 322 s:5′-ccgccgcgacuucgccgagau-3′ human  18 ± 2 SEQ ID NO: 309 as:3′-auggcggcgcugaagcggcucua-5′ SEQ ID NO: 310 B333 333 s:5′-ucgccgagauguccagccagc-3′ human  18 ± 5 SEQ ID NO: 311 as:3′-gaagcggcucuacaggucggucg-5′ SEQ ID NO: 312 B450 450 s:5′-uugaguucgguggggucaugu-3′ human +  17 ± 1 SEQ ID NO: 313 as:3′-gaaacucaagccaccccaguaca-5′ mouse SEQ ID NO: 314 B83 83 s:5′-gagugggaugcgggagaugug-3′ human  17 ± 3 SEQ ID NO: 315 as:3′-ugcucacccuacgcccucuacac-5′ SEQ ID NO: 316 B582 582 s:5′-gggaugccuuuguggaacugu-3′ human  17 ± 3 SEQ ID NO: 317 as:3′-gacccuacggaaacaccuugaca-5′ SEQ ID NO: 318 B658 658 s:5′-gcucaguuuggcccugguggg-3′ human α  16 ± 5 SEQ ID NO: 319 as:3′-gacgagucaaaccgggaccaccc-5′ SEQ ID NO: 320 B80 80 s:5′-uacgagugggaugcgggagau-3′ human  16 ± 4 SEQ ID NO: 321 as:3′-cgaugcucacccuacgcccucua-5 SEQ ID NO: 322 B130 130 s:5′-cgcaccgggcaucuucuccuc-3′ human  15 ± 2 SEQ ID NO: 323 as:3′-gggcguggcccguagaagaggag-5′ SEQ ID NO: 324 B294 294 s:5′-aggccggcgacgacuucuccc-3′ human  15 ± 2 SEQ ID NO: 325 as:3′-gguccggccgcugcugaagaggg-5′ SEQ ID NO: 326 B686 686 s:5′-aucacccugggugccuaucug-3′ human α  15 ± 3 SEQ ID NO: 327 as:3′-cguagugggacccacggauagac-5′ SEQ ID NO: 328 B292 292 s:5′-ccaggccggcgacgacuucuc-3′ human  15 ± 4 SEQ ID NO: 329 as:3′-gcgguccggccgcugcugaagag-5′ SEQ ID NO: 330 B291 291 s:5′-gccaggccggcgacgacuucu-3′ human  15 ± 3 SEQ ID NO: 331 as:3′-ggcgguccggccgcugcugaaga-5′ SEQ ID NO: 332 B356 356 s:5′-caccugacgcccuucaccgcg-3′ human +  14 ± 4 SEQ ID NO: 333 as:3′-acguggacugcgggaaguggcgc-5′ mouse SEQ ID NO: 334 B663 663 s:5′-guuuggcccuggugggagcuu-3′ human α  14 ± 3 SEQ ID NO: 335 as:3′-gucaaaccgggaccacccucgaa-5′ SEQ ID NO: 336 B586 586 s:5′-ugccuuuguggaacuguacgg-3′ human  14 ± 2 SEQ ID NO: 337 as:3′-cuacggaaacaccuugacaugcc-5′ SEQ ID NO: 338 B353 353 s:5′-cugcaccugacgcccuucacc-3′ human +  13 ± 1 SEQ ID NO: 339 as:3′-ucgacguggacugcgggaagugg-5′ mouse SEQ ID NO: 340 B512 512 s:5′-aacaucgcccuguggaugacu-3′ human +  13 ± 1 SEQ ID NO: 341 as:3′-uguuguagcgggacaccuacuga-5′ mouse SEQ ID NO: 342 B657 657 s:5′-ugcucaguuuggcccuggugg-3′ humanα  13 ± 1 SEQ ID NO: 343 as:3′-agacgacucaaaccgggaccacc-5′ SEQ ID NO: 344 B473 473 s:5′-guggagagcgucaaccgggag-3′ human  13 ± 4 SEQ ID NO: 345 as:3′-cacaccucucgcaguuggcccuc-5′ SEQ ID NO: 346 B532 532 s:5′-ugaguaccugaaccggcaccu-3′ human  13 ± 1 SEQ ID NO: 347 as:3′-ugacucauggacuuggccgugga-5′ SEQ ID NO: 348 B504 504 s:5′-ugguggacaacaucgcccugu-3′ human +  12 ± 3 SEQ ID NO: 349 as:3′-ggaccaccuguuguagcgggaca-5′ mouse SEQ ID NO: 350 B382 382 s:5′-acgcuuugccacgguggugga-3′ human +  12 ± 2 SEQ ID NO: 351 as:3′-ccugcgaaacggugccaccaccu-5′ mouse SEQ ID NO: 352 B355 355 s:5′-gcaccugacgcccuucaccgc-3′ human +  11 ± 4 SEQ ID NO: 353 as:3′-gacguggacugcgggaaguggcg-5′ mouse SEQ ID NO: 354 B673 673 s:5′-ggugggagcuugcaucacccu-3′ human α  11 ± 1 SEQ ID NO: 355 as:3′-gaccacccucgaacguaguggga-5′ SEQ ID NO: 356 B561 561 s:5′-ggauccaggauaacggaggcu-3′ human +  10 ± 2 SEQ ID NO: 357 as:3′-gaccuagguccuauugccuccga-5′ mouse SEQ ID NO: 358 B16 16 s:5′-aacaggguacgauaaccggga-3′ human   9 ± 3 SEQ ID NO: 359 as:3′-ucuugucccaugcuauuggcccu-5′ SEQ ID NO: 360 B568 568 s:5′-ggauaacggaggcugggaugc-3′ humanα +   9 ± 1 SEQ ID NO: 361 as:3′-guccuauugccuccgacccuacg-5′ mouse SEQ ID NO: 362 B664 664 s:5′-uuuggcccuggugggagcuug-3′ human α   9 ± 3 SEQ ID NO: 363 as:3′-ucaaaccgggaccacccucgaac-5′ SEQ ID NO: 364 B15 15 s:5′-gaacaggguacgauaaccggg-3′ human   8 ± 2 SEQ ID NO: 365 as:3′-cucaagacccaugcuauuggccc-5′ SEQ ID NO: 366 B354 354 s:5′-ugcaccugacgcccuucaccg-3′ human +   8 ± 3 SEQ ID NO: 367 as:3′-cgacguggacugcgggaaguggc-5′ mouse SEQ ID NO: 368 B664 664 s:5′-uuuggcccuggugggagcuug-3′ human α   8 ± 5 SEQ ID NO: 369 as:3′-ucaaaccgggaccacccucgaac-5′ SEQ ID NO: 370 B17 17 s:5′-acaggguacgauaaccgggag-3′ human   7 ± 0 SEQ ID NO: 371 as:3′-cuugucccaugcuauuggcccuc-5′ SEQ ID NO: 372 B293 293 s:5′-caggccggcgacgacuucucc-3′ human   7 ± 1 SEQ ID NO: 373 as:3′-cgguccggccgcugcugaagagg-5′ SEQ ID NO: 374 B296 296 s:5′-gccggcgacgacuucucccgc-3′ human   7 ± 3 SEQ ID NO: 375 as:3′-uucggccgcugcugaagagggcg-5′ SEQ ID NO: 376 B303 303 s:5′-acgacuucucccgccgcuacc-3′ human   7 ± 2 SEQ ID NO: 377 as:3′-gcugcugaagagggcggcgaugg-5′ SEQ ID NO: 378 B455 455 s:5′-uucgguggggucaugugugug-3′ human +   7 ± 2 SEQ ID NO: 379 as:3′-ucaagccaccccaguacacacac-5′ mouse SEQ ID NO: 380 B8 8 s:5′-gcugggagaacaggguacgac-3′ human   7 ± 3 SEQ ID NO: 381 as:3′-ugcgacccucuugucccaugcug-5′ SEQ ID NO: 382 B129 129 s:5′-ccgcaccgggcaucuucuccu-3′ human   6 ± 3 SEQ ID NO: 383 as:3′-ggggcguggcccguagaagagga-5′ SEQ ID NO: 384 B304 304 s:5′-cgacuucucccgccgcuaccg-3′ human   6 ± 2 SEQ ID NO: 389 as:3′-cugcugaagagggcggcgauggc-5′ SEQ ID NO: 390 B682 682 s:5′-uugcaucacccugggugccua-3′ human α   6 ± 4 SEQ ID NO: 385 as:3′-cgaacguagugggacccacggau-5′ SEQ ID NO: 386 B682 682 s:5′-uugcaucacccugggugccua-3′ human α   6 ± 4 SEQ ID NO: 387 as:3′-cgaacguagugggacccacggau-5′ SEQ ID NO: 388 B506 506 s:5′-guggacaacaucgcccugugg-3′ human +   5 ± 6 SEQ ID NO: 391 as:3′-accaccuguuguagcgggacacc-5′ mouse SEQ ID NO: 392 B138 138 s:5′-gcaucuucuccucccagcccg-3′ human   4 ± 2 SEQ ID NO: 393 as:3′-cccguagaagaggagggucgggc-5′ SEQ ID NO: 394 B385 385 s:5′-cuuugccacggugguggagga-3′ human +   4 ± 2 SEQ ID NO: 395 as:3′-gcgaaacggugccaccaccuccu-5′ mouse SEQ ID NO: 396 B131 131 s:5′-gcaccgggcaucuucuccucc-3′ human   3 ± 6 SEQ ID NO: 397 as:3′-ggcguggcccguagaagaggagg-5′ SEQ ID NO: 398 B600 600 s:5′-uguacggccccagcaugcggc-3′ human α   3 ± 1 SEQ ID NO: 399 as:3′-ugacaugccggggucguacgccg-5′ SEQ ID NO: 400 B653 653 s:5′-acucugcucaguuuggcccug-3′ humanα   3 ± 4 SEQ ID NO: 401 as:3′-ucugagacgagucaaaccgggac-5′ SEQ ID NO: 402 B665 665 s:5′-uuggcccuggugggagcuugc-3′ human α   3 ± 10 SEQ ID NO: 403 as:3′-caaaccgggaccacccucgaacg-5′ SEQ ID NO: 404 B666 666 s:5′-uggcccuggugggagcuugca-3′ humanα   3 ± 3 SEQ ID NO: 405 as:3′-aaaccgggaccacccucgaacgu-5′ SEQ ID NO: 406 B684 684 s:5′-gcaucacccugggugccuauc-3′ human α   3 ± 1 SEQ ID NO: 407 as:3′-aacguagugggacccacggauag-5′ SEQ ID NO: 408 B672 672 s:5′-uggugggagcuugcaucaccc-3′ human α   2 ± 2 SEQ ID NO: 409 as:3′-ggaccacccucgaacguaguggg-5′ SEQ ID NO: 410 B602 602 s:5′-uacggccccagcaugcggccu-3′ humanα   2 ± 3 SEQ ID NO: 411 as:3′-acaugccggggucguacgccgga-5′ SEQ ID NO: 412 B581 581 s:5′-ugggaugccuuuguggaacug-3′ human   2 ± 6 SEQ ID NO: 413 as:3′-cgacccuacggaaacaccuugac-5′ SEQ ID NO: 414 B14 14 s:5′-agaacaggguacgauaaccgg-3′ human   1 ± 3 SEQ ID NO: 415 as:3′-ccucuugucccaugcuauuggcc-5′ SEQ ID NO: 416 B305 305 s:5′-gacuucucccgccgcuaccgc-3′ human   1 ± 2 SEQ ID NO: 417 as:3′-ugcugaagagggcggcgauggcg-5′ SEQ ID NO: 418 B651 651 s:5′-agacucugcucaguuuggccc-3′ humanα   1 ± 4 SEQ ID NO: 419 as:3′-cuucugagacgagucaaaccggg-5′ SEQ ID NO: 420 B675 675 s:5′-ugggagcuugcaucacccugg-3′ human α   0 ± 11 SEQ ID NO: 421 as:3′-ccacccucgaacguagugggacc-5′ SEQ ID NO: 422 B674 674 s:5′-gugggagcuugcaucacccug-3′ human α   0 ± 1 SEQ ID NO: 423 as:3′-accacccucgaacguagugggac-5′ SEQ ID NO: 424 B290 290 s:5′-cgccaggccggcgacgacuuc-3′ human   0 ± 2 SEQ ID NO: 425 as:3′-aggcgguccggccgcugcugaag-5′ SEQ ID NO: 426 B73 73 s:5′-gaggggcuacgagugggaugc-3′ human +  −1 ± 1 SEQ ID NO: 427 as:3′-gucuccccgaugcucacccuacg-5′ mouse SEQ ID NO: 428 B162 162 s:5′-acacgccccauccagccgcau-3′ human  −5 ± 1 SEQ ID NO: 429 as:3′-cgugugcgggguaggucggcgua-5′ SEQ ID NO: 430 B679 679 s:5′-agcuugcaucacccugggugc-3′ human α  −6 ± 1 SEQ ID NO: 431 as:3′-ccucgaacguagugggacccacg-5′ SEQ ID NO: 432 B71 71 s:5′-cagaggggcuacgagugggau-3′ human  −6 ± 5 SEQ ID NO: 433 as:3′-gcgucuccccgaugcucacccua-5′ SEQ ID NO: 434 B599 599 s:5′-cuguacggccccagcaugcgg-3′ humanα  −7 ± 7 SEQ ID NO: 435 as:3′-uugacaugccggggucguacgcc-5′ SEQ ID NO: 436 B681 681 s:5′-cuugcaucacccugggugccu-3′ human α  −7 ± 1 SEQ ID NO: 437 as:3′-ucgaacguagugggacccacgga-5′ SEQ ID NO: 438 B683 683 s:5′-ugcaucacccugggugccuau-3′ human α  −10 ± 4 SEQ ID NO: 439 as:3′-gaacguagugggacccacggaua-5′ SEQ ID NO: 440 B691 691 s:5′-ccugggugccuaucugggcca-3′ humanα  −10 ± 6 SEQ ID NO: 441 as:3′-ugggacccacggauagacccggu-5′ SEQ ID NO: 442 B58 58 s:5′-uuauaagcugucgcagagggg-3′ human  −16 ± 5 SEQ ID NO: 443 as:3′-guaauauucgacagcgucucccc-5′ SEQ ID NO: 444 K4 2606 of s:5′-gaugaggaucguuucgcauga-3′ n.a.   0 SEQ ID NO: 445 negative U55763 as:3′-uccuacuccuagcaaagcguacu-5′ SEQ ID NO: 446 control

Example 2 Optimization of siRNAs by Chemical Modification

As has been experienced by those working in the antisense field,ribonucleic acids are often quickly degraded by a range of nucleasespresent in virtually all biological environments, e.g. endonucleases,exonucleases etc. This vulnerability may be circumvented by chemicallymodifying these oligonucleotides such that nucleases may no longerattack. Consequentially, 8 siRNAs were chosen, designated B21, B22, B25,B133, B442, B519, B522, B523, and B529 in Table 2, which showed superioractivity in the assay described in Example 1, for the testing of theeffect of stabilizing modifications on the activity of siRNAs to inhibitgene expression.

To establish whether the chemical modification of nucleotides interfereswith the ability of siRNAs to inhibit gene expression, we chose to startwith a minimal modification. siRNAs corresponding to B21, B22, B25,B133, B442, B519, B522, B523, and B529, but comprising 2′-O-Methylsubstituted nucleotides in positions 21 and 22 (counting 5′ to 3′) ofthe antisense strands were synthesized and their activity was tested inKB-GFP-BCL2 cells as described in Example 1 above (B529-2OMe, B25-2OMe,B21-2OMe, B22-2OMe, B522-2OMe, B523-2OMe, B519-2OMe, B133-2OMe,B442-2OMe).

TABLE 3 List of siRNAs employed in testing the influence of 2′-O-Methylnucleotide modifications on siRNA efficacy (spaces inserted insense-strand sequences to show alignment) Efficacy of 5′-Startunmodified Efficacy Name nucleotide Sequence [%] [%] Seq. ID B529- 529s: 5′-gacugaguaccugaaccggca-3′ 81 ± 0 78 ± 4 (SEQ ID NO: 447 2OMe as:3′-uamcmugacucauggacuuggccgu-5′ (SEQ ID NO: 448 B25-  25 s:5′-cgauaaccgggagauagugau-3′ 80 ± 1 74 ± 4 (SEQ ID NO: 449 2OMe as:3′-aumgmcuauuggcccucuaucacua-5′ (SEQ ID NO: 450 B21-  21 s:5′-gguacgauaaccgggagauag-3′ 79 ± 2 82 ± 0 (SEQ ID NO: 451 2OMe as:3′-ccmcmcaugcuauuggcccucuauc-5′ (SEQ ID NO: 452 B22-  22 s:5′-guacgauaaccgggagauagu-3′ 79 ± 1 83 ± 2 (SEQ ID NO: 453 2OMe as:3′-ccmcmaugcuauuggcccucuauca-5′ (SEQ ID NO: 454 B522- 522 s:5′-uguggaugacugaguaccuga-3′ 79 ± 3 73 ± 3 (SEQ ID NO: 455 2OMe as:3′-ggmamcaccuacugacucauggacu-5′ (SEQ ID NO: 456 B523- 523 s:5′-guggaugacugaguaccugaa-3′ 75 ± 2 80 ± 2 (SEQ ID NO: 457 2OMe as:3′-gamcmaccuacugacucauggacuu-5′ (SEQ ID NO: 458 B519- 519 s:5′-cccuguggaugacugaguacc-3′ 73 ± 0 65 ± 3 (SEQ ID NO: 459 2OMe as:3′-gcmgmggacaccuacugacucaugg-5′ (SEQ ID NO: 460 B133- 133 s:5′-accgggcaucuucuccuccca-3′ 70 ± 1 66 ± 10 (SEQ ID NO: 461 2OMe as:3′-cgmumggcccguagaagaggagggu-5′ (SEQ ID NO: 462 B442- 442 s:5′-gugccuucuuugaguucggugg-3′ 70 ± 5 72 ± 0 (SEQ ID NO: 463 2OMe as:3′-camcmcggaagaaacucaagccacc-5′ (SEQ ID NO: 464

When comparing the efficacy of inhibition of the unmodified siRNAs(4^(th) column in Table 3) to those of the siRNAs comprising the 22′-O-Methyl modifications (5^(th) column in Table 3), it is evident thatthe modifications had only a minor effect on efficacy, often withinerror limits of the assay.

Another modification often employed in attempting to modify theproperties of oligonucleotides, e.g. increase plasma binding, is theintroduction of phosphorothioate linkages into the backbone of theoligonucleotide. To test the efficacy of phosphorothioate-modifiedsiRNAs, we synthesized siRNAs wherein the phosphodiester linkagesbetween positions 21 and 22, and 22 and 23, respectively, (counting 5′to 3′) of the antisense strands of B529, B25, B519, and B442 werereplaced by phosphorothioate linkages (B529-2PO, B25-2PO, B519-2PO,B442-2PO). In addition, one siRNA was synthesized wherein thephosphodiester linkages between positions 1 and 2, 2 and 3, 3 and 4, 4and 5, 19 and 20, 20 and 21, 21 and 22, and 22 and 23 of the antisensestrand, and 1 and 2, 2 and 3, 3 and 4, 4 and 5, 17 and 18, 18 and 19, 19and 20, and 20 and 21 of the sense strand, respectively, (counting 5′ to3′) of B442 were replaced by phosphorothioate linkages (B442-16PO).

TABLE 4 List of siRNAs employed in testing the influence of 2′-O-Methylnucleotide modifications on siRNA efficacy Efficacy of Effi- 5′-Startunmodified cacy Name nucleotide Sequence [%] [%] Seq. ID B529- 529 s:5′-gacugaguaccugaaccggca-3′ 81 ± 0 79 ± 2 SEQ ID NO: 465 2PO as:3′-uacugacucauggacuuggccgu-5′ SEQ ID NO: 466 B25-  25 s:5′-cgauaaccgggagauagugau-3′ 80 ± 1 75 ± 2 SEQ ID NO: 467 2PO as:3′-augcuauuggcccucuaucacua-5′ SEQ ID NO: 468 B519- 519 s:5′-cccuguggaugacugaguacc-3′ 73 ± 0 70 ± 2 SEQ ID NO: 469 2PO as:3′-gcgggacaccuacugacucaugg-5′ SEQ ID NO: 470 B442- 442 s:5′-ggccuucuuugaguucggugg-3′ 70 ± 5 75 ± 3 SEQ ID NO: 471 2PO as:3′-caccggaagaaacucaagccacc-5′ SEQ ID NO: 472 B442- 442 s:5′-ggccuucuuugaguucggugg-3′ 70 ± 5 72 ± 4 SEQ ID NO: 473 16PO as:3′-caccggaagaaacucaagccacc-5′ SEQ ID NO: 474

Again, the replacement of phosphodiester linkages by phosphorothioatelinkages had no significant effect on the efficacy of these siRNAs.

Next, we examined the effect of lipophilic groups linked to the 3′- or5′-end of the sense and/or antisense strand of siRNAs. Lipophilic groupstethered to an oligonucleotide have been held to increase the permeationof oligonucleotides through the membrane of cells even in the absence ofagents that aid transfection. We tested a 12-dodecanoic acidbisdecylamide group linked to the 5′-end of the sense strand, the 5′-endof the antisense strand, and to the 5′ end of both strands, of B442-2OMe(B442-5′C32s, B442-5′C32 as, B442-5′C32b). Furthermore, we tested acholesteryl derivative linked via a phosphorothioate linkage to the3′-end of the sense strand of B442-2OMe, combined with additionalreplacement of the phosphodiester linkages between positions 21 and 22,and 22 and 23, respectively, (counting 5′ to 3′) of the antisense strand(B442-3′Chol).

TABLE 5 List of siRNAs employed in testing the influence of lipophilicgroups linked to the 3′- or 5′-end of the sense and/or antisense strandon siRNA efficacy (spaces inserted in sense-strand sequences to showalignment) Efficacy of B442- 5′-Start 2OMe Efficacy Name nucleotideSequence [%] [%] Seq. ID B442- 442 a: 5′-C32-g gccuucuuugaguucggugg-3′72 ± 0  57 ± 3 SEQ ID NO: 475 5′C32s as: 3′-camcmcggaagaaacucaagccacc-5′SEQ ID NO: 476 B442- 442 s: 5′-g gccuucuuugaguucggugg-3′ 72 ± 0  68 ± 3SEQ ID NO: 477 5′C32as as: 3′-camcmcggaagaaacucaagccacc-C32-5′ SEQ IDNO: 478 B442- 442 s: 5′-C32-g gccuucuuugaguucggugg-3′ 72 ± 0 −23 ± 5 SEQID NO: 479 5′C32b as: 3′-camcmcggaagaaacucaagccacc-C32-5′ SEQ ID NO: 480B442- 442 s: 5′-g gccuucuuugaguucggugg-Chol-3′ 72 ± 0  54 ± 2 SEQ ID NO:481 3′Chol as: 3′-camcmcggaagaaacucaagccacc-5′ SEQ ID NO: 482

As evident from Table 5, the lipophilic 12-dodecanoic acid bisdecylamidegroup had virtually no effect on gene inhibition efficacy when linked tothe 5′-end of the antisense strand, slightly reduced efficacy whenlinked to the 5′-end of the sense strand, and abolished activity whenlinked to the 5′-end of both strands. Linking a cholesteryl derivativeto the 3′-end of the sense strand and replacing two phosphodiesterlinkages on the 3′-end of the antisense strand with phosphorothioatelinkages only slightly reduced activity.

Example 3 Inhibition of Bcl-2 Gene Expression by RNA Interference inMelJuso Cells

To further test the ability of siRNAs bearing nucleotide modificationsto inhibit the expression of Bcl-2, a range of differently modifiedsiRNAs specific for Bcl-2, and derived from siRNA B442 above, was testedin MelJuso cells stably transfected with a Bcl-2/GFP-fusion protein. ThesiRNAs tested bore various combinations of the following modifications:2′-O-Methyl groups in positions 21 and 22 (counting 5′ to 3′) of theantisense strand; phosphorothioate linkages between positions 21 and 22(counting 5′ to 3′) of the antisense strand; phosphorothioate linkagesbetween the 4 3′-most and the 4 5′-most nucleotides in the sense strand;all linkages replaced by phosphorothioates in the sense or antisensestrand; and a cholesteryl derivative linked to the 5′-end of the senseor antisense strand via a phosphorothioate linkage.

Creation of Cell Line Mel Juso-GFP-BCL2

A reporter cell line for estimating the efficacy of siRNAs in inhibitingthe expression of BCL2 is constructed by transfecting Mel Juso cells(DSMZ No ACC 74) with a reporter construct from which an mRNA istranscribed encoding an eGFP-BCL2 fusion protein. The efficacy ofinhibition may be measured in such cells by comparing the fluorescenceintensity of eGFP in such cells after treatment with a siRNA comprisinga BCL2 sequence with the fluorescence intensity in such cells treatedwith a control siRNA.

The open reading frame of human BCL-2 (alpha splice form, GenBankaccession number M13994) is PCR-amplified from a human BD™Marathon-Ready cDNA library (BD Biosciences Clontech, Palo Alto, Calif.,USA, Cat. #: 639343) using the BD Advantage HF 2 PCR kit (BD BiosciencesClontech, Palo Alto, Calif., USA Cat. #: 639123). Primer, nucleotide andenzyme concentration are used according to manufacturer's instructions.Amplification is performed in 30 cycles with the following three stepsin each cycle: 20 sec. 95° C., 30 sec. 62° C., 60 sec. 72° C. A finalstep of 120 sec at 72° C. terminates the amplification reaction. Primersare AAA CTC GAG GCG CAC GCT GGG AGA ACG GGG (SEQ ID NO:490) (introducinga XhoI (italics) restriction site upstream of the codon coding for aminoacid 2 of BCL2) and AAA TCT AGA TCA CTT GTG GCT CAG ATA GGC (SEQ IDNO:491) (introducing a XbaI restriction site (italics) after the BCL2stop codon (double underlined)). The PCR product is gel-purified on a0.8% agarose gel, digested with XhoI and XbaI and ligated into pEGFP-C3(BD Biosciences Clontech, Palo Alto Calif., USA; Cat. #: 632315)digested with XhoI and XbaI. The correct insertion of the cDNA isverified by sequencing. The plasmid is transfected into Mel Juso cellsby lipofection with FuGene6 (Roche Cat No. 1814443) andNeomycin-resistant and fluorescing clones are identified in three roundsof: selection in the presence of G418 for 48 h followed by selection offluorescent cells and replating of single cells using FACS-analysis.

Introduction of siRNAs into Mel Juso-GFP-BCL2-cells and Determination ofGene Expression Inhibition by FACS Analysis

Mel Juso-GFP-BCL2-Cells (about 80% confluent) were trypsinized from 96mm Petri dishes with 5 ml trypsin-EDTA (0.25% Trypsin; 1 mM Na-4-EDTA;Gibco/Invitrogen, Karlsruhe, Germany) for 3-5 min at 37° C. 5 ml cellculture medium (DMEM supplemented with 10% FCS and 2500 μg/ml Neomycin,all GBCO BRL, Paisley, UK) were added and cells were centrifuged at 400g for 5 minutes at room temperature. The cell pellet was resuspended in250 μl cell culture medium and the cell number per unit volumedetermined in a Neubauer chamber. The resuspended cells were diluted toa density of 50 000 cells per ml cell culture medium and 2 ml of thissuspension were plated into one well of a 6 well plate. Mel Juso cellswere seeded 24 h before siRNA treatment to allow adherent cell growth.After 24 h culture medium was removed and cultures were incubated for 4h with 50 nM siRNA pre-complexed in Opti-MEM medium with Oligofectamin(both from Invitrogen, Carlsbad, USA) according to the manufacturer'sprotocol. After incubation, the incubation medium was replaced bycomplete medium and cells were cultivated under standard conditions 37°C. and 5% CO2 for 72 h (Heracell incubator, Kendro Laboratory productsGmbH, Langenselbold, Germany).

Cells were harvested by removing the cell culture medium, adding 500 μltrypsin-EDTA (Gibco-Invitrogen GmbH, Karlsruhe, Germany) per well andincubating for 3-5 min at 37° C. 1 ml cell culture medium was added tothe trypsin solution and the cells were resuspended. The suspension wastransferred to FACS tubes (5 ml, Sarstedt AG & Co., Nümbrecht, Germany)and centrifuged at 400 g for 5 min. Pellets were resuspended in 1 ml PBS(Biochrom, Cambridge, UK) and eGFP-fluorescence was measured byflow-cytometry (FACS Calibur, Becton Dickinson, Franklin Lakes, N.J.,USA) 10.000 cells were counted per tube.

TABLE 6 List of siRNAs employed in testing the influence of variousmodifications of the sense and/or antisense strand on siRNA efficacy inMelJuso cells, and the efficiency of a cholesteryl derivative tetheredto one or both strands in facilitating cell entry of siRNAs withouttransfection aid Effi-cacy Name Lipofection Sequence [%] Seq. ID AL-DUP-X 5′-ggccuucuuugaguucggugg-3′  49 ± 6% SEQ ID NO: 483 51085′-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 484 AL-DUP- X5′-ggccuucuuugaguucggugg-3′  51 ± 14% SEQ ID NO: 485 51095′-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 486 AL-DUP- X5′-ggccuucuuugaguucggugg-3′  46 ± 12% SEQ ID NO: 485 51105′-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 484 AL-DUP- X5′-Chol-ggccuucuuugaguucggugg-3′  44 ± 3% SEQ ID NO: 487 51115′-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 486 AL-DUP- X5′-Chol-ggccuucuuugaguucggugg-3′   3 ± 2% SEQ ID NO: 488 51125′-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 486 AL-DUP- X5′-Chol-ggccuucuuugaguucggugg-3′ −30 ± 14% SEQ ID NO: 488 51135′-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 484 AL-DUP- X5′-ggccuucuuugaguucggugg-3′ −23 ± 1% SEQ ID NO: 483 51145′-Chol-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 489 AL-DUP- X5′-Chol-ggccuucuuugaguucggugg-3′  23 ± 3% SEQ ID NO: 487 51155′-Chol-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 489 AL-DUP- X5′-Chol-ggccuucuuugaguucggugg-3′  32 ± 2% SEQ ID NO: 488 51165′-Chol-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 489 AL-DUP- X5′-ggccuucuuugaguucggugg-3′ −22 ± 2% SEQ ID NO: 485 51175′-Chol-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 489 AL-DUP- X5′-ggccuucuuugaguucggugg-3′  66 ± 11% SEQ ID NO: 483 51215′-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 486 AL-DUP-5′-ggccuucuuugaguucggugg-3′  −2 ± 3% SEQ ID NO: 485 51105′-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 484 AL-DUP-5′-Chol-ggccuucuuugaguucggugg-3′  17 ± 1% SEQ ID NO: 488 51125′-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 486 AL-DUP-5′-Chol-ggccuucuuugaguucggugg-3′  15 ± 2% SEQ ID NO: 488 51135′-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 484 AL-DUP-5′-ggccuucuuugaguucggugg-3′  13 ± 1% SEQ ID NO: 483 51145′-Chol-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 489 AL-DUP-5′-Chol-ggccuucuuugaguucggugg-3′  13 ± 2% SEQ ID NO: 487 51155′-Chol-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 489 AL-DUP-5′-Chol-ggccuucuuugaguucggugg-3′  16 ± 1% SEQ ID NO: 485 51165′-Chol-ccaccgaacucaaagaaggccmamc-3′ SEQ ID NO: 489

As is evident from Table 6, the modification of nucleotides withphosphorothioate linkages in the backbone was generally well tolerated,as were 2′-O-methyl modifications of nucleotides located near the3′-terminus of the sense strand. The results obtained using this assaywith cholesteryl-derivative modified siRNAs AL-DUP-5112 throughAL-DUP-5117 are somewhat inconclusive when lipofection is employed tofacilitate entry of the siRNA into the cell. However, we have observedin the past that lipofection agents in connection with phosphorothioate-and/or cholesteryl-modified oligonucleotides can sometimes give rise toexperimental artifacts (data not shown). It should therefore not beconcluded that the 5′-cholesteryl modification abolishes siRNA activity.In addition, from those results obtained with the same siRNAs in theabsence of lipofection agent, it is clear that the cholesterylmodification allows entry of the siRNAs into cells in a setting that ismore relevant to the situation encountered within the body of an animal,e.g. a human, namely the absence of high concentrations of a lipofectionagent. AL-DUP-5110, which is identical to AL-DUP-5114 except for thecholesteryl-derivative ligand, did not have any effect when incubatedwith cells without lipofection agent, while AL-DUP-5114 inhibited bcl-2expression under these circumstances.

Example 4 Inhibition of Bcl-2 Gene Expression by RNA Interference inHuman Pancreatic Cancer YAP C Cells

The cells of the human pancreatic Yap C cancer line (GermanMicroorganism and Cell Culture Collection, Braunschweig, (No. ACC 382)),were cultured at 37° C., 5% CO₂ in RPMI 1640 medium (Biochrom Corp.,Berlin) with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin.Human skin fibroblasts were cultured under the same conditions inDulbecco's MEM with 10% FCS and 1% penicillin/streptomycin.

The double-stranded oligoribonucleotides used for transfection have thefollowing sequences, designated as SEQ ID No:1 to SEQ ID No:6 in thesequence protocol:

dsRNA 1, which is complementary to a first sequence of the human Bcl-2gene: S2: 5′-caggaccucgccgcugcagacc-3′ (SEQ ID NO: 1) S1:3′-cgguccuggagcggcgacgucugg-5′ (SEQ ID NO: 2) dsRNA 2, which iscomplementary to a second sequence of the human Bcl-2 gene: S2:5′-gccuuuguggaacuguacggcc-3′ (SEQ ID NO: 3) S1:3′-uacggaaacaccuugacaugccgg-5′ (SEQ ID NO: 4) dsRNA 3, which iscomplementary to a sequence of the neomycin resistance gene: S2:5′-caaggaugaggaucguuucgca-3′ (SEQ ID NO: 5) S1:3′-ucuguccuacuccuagcaaagcg-5′ (SEQ ID NO: 6)

Transfection was carried out in a 6-well plate with oligofectamine(Invitrogen Corp., Karlsruhe). 250,000 cells were placed in each well.Transfection of the double-stranded oligoribonucleotides was carried outin accordance with the oligofectamine protocol recommended by Invitrogen(the data relate to 1 well of a 6-well plate):

10 μl of the double-stranded oligoribonucleotides (0.1-10 μM) werediluted with 175 μl cell culture medium without additives. 3 μloligofectamine were diluted with 12 μl cell culture medium withoutadditives, and incubated for 10 minutes at room temperature. The dilutedoligofectamine was then added to the diluted double-strandedoligoribonucleotides, mixed, and incubated for 20 minutes at roomtemperature. During this time, the cells to be transfected were washedonce with cell culture medium without additives, and 800 μl of freshcell culture medium was added so that the transfection end volume was1000 μl. This results in a double-stranded oligoribonucleotide endconcentration of 1-100 μM. The transfection media was incubated with thecells for four hours at 37′IC. 500 μl of cell culture medium with 30%FCS were then placed in each well, i.e. final concentration of FCS was10%. The cells were then incubated for 120 hours at 37° C., at whichtime they were washed with phosphate buffered saline (PBS), trypsinizedand centrifuged for 10 minutes at 100 g. The supernatant fluid wasdiscarded, and the pellet was incubated in the dark with hypotonicpropidium iodide solution for 30 minutes at 4° C. The pelletted cellswere then analyzed by flow cytometry using a FACSCaliburfluorescence-activated cell sorter (BD GmbH, Heidelberg).

Both the double-stranded oligoribonucleotides dsRNA 1 and dsRNA 2decreased the inhibition of apoptosis mediated by Bcl-2 in the humanpancreatic cancer cells studied. No additional stimulation of apoptosiswas required to induce or initiate apoptosis. The apoptosis rate roseindependent of incubation time. FIG. 1 shows the result achieved withdsRNA 1 and FIG. 2 that with dsRNA 2. Whereas untreated YAP C controlcells and cells with which the described methods of transfection werecarried out without double-stranded oligoribonucleotides(mock-transfected cells) showed an apoptosis rate of only 3.8% and 7.1%after 120 hours incubation, the apoptosis rate achieved with 100 nMdsRNA rose to 37.2% for transfection with dsRNA 1 and 28.9% fortransfection with dsRNA 2. Control transfection with dsRNA 3 led to amaximum apoptosis rate of 13.5%. This represents no significant increasewhen compared to mock-transfected cells, and proves the sequencespecificity of the action of the dsRNA 1 and dsRNA 2. As a control, skinfibroblasts were transfected as non-transformed cells with dsRNA 1 anddsRNA 2. After 120 hours, these cells showed no significant increase inapoptosis rate.

Example 5 Treatment of a Pancreatic Cancer Patient with dsRNA 1 and 2

In this Example, dsRNA 1 and 2 are injected into a pancreatic cancerpatient and shown to specifically inhibit Bcl-2 gene expression.

Synthesis and Preparation of dsRNAs

dsRNA 1 and 2 directed against the Bcl-2 gene are chemically synthesizedwith or without a hexaethylene glycol linker. Oligoribonucleotides aresynthesized with an RNA synthesizer (Expedite 8909, Applied Biosystems,Weiterstadt, Germany) and purified by High Pressure LiquidChromatography (HPLC) using NucleoPac PA-100 columns, 9×250 mm (DionexCorp.; low salt buffer: 20 mM Tris, 10 mM NaClO4, pH 6.8, 10%acetonitrile; the high-salt buffer was: 20 mM Tris, 400 mM NaClO4, pH6.8, 10% acetonitrile. flow rate: 3 ml/min). Formation ofdouble-stranded dsRNAs is then achieved by heating a stoichiometricmixture of the individual antisense strands (10 μM) in 10 mM sodiumphosphate buffer, pH 6.8, 100 mM NaCl, to 80-90° C., with subsequentslow cooling to room temperature over 6 hours.

In addition, dsRNA molecules with linkers may be produced by solid phasesynthesis and addition of hexaethylene glycol as a non-nucleotide linker(Jeremy, D., et al., Biochem. (1996), 35:14665-14670). A hexaethyleneglycol linker phosphoramidite (Chruachem Ltd, Todd Campus, West ofScotland Science Park, Acre Road, Glasgow, G20 OUA, Scotland, UK) iscoupled to the support bound oligoribonucleotide employing the samesynthetic cycle as for standard nucleoside phosphoramidites (ProligoBiochemie GmbH, Georg-Hyken-Str. 14, Hamburg, Germany) but withprolonged coupling times. Incorporation of linker phosphoramidite iscomparable to the incorporation of nucleoside phosphoramidites.

dsRNA Administration and Dosage

The present example provides for pharmaceutical compositions for thetreatment of human pancreatic cancer patients comprising atherapeutically effective amount of a dsRNA 1 and dsRNA 2 as disclosedherein, in combination with a pharmaceutically acceptable carrier orexcipient. dsRNAs useful according to the invention may be formulatedfor oral or parenteral administration. The pharmaceutical compositionsmay be administered in any effective, convenient manner including, forinstance, administration by topical, oral, anal, vaginal, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes among others. One of skill in the art can readily prepare dsRNAsfor injection using such carriers that include, but are not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. Additional examples of suitable carriers are foundin standard pharmaceutical texts, e.g. “Remington's PharmaceuticalSciences”, 16th edition, Mack Publishing Company, Easton, Pa. (1980).

RNA Purification and Analysis

Efficacy of the dsRNA treatment is determined at defined intervals afterthe initiation of treatment using real time PCR on total RNA extractedfrom tissue biopsies. Cytoplasmic RNA from tissue biopsies, taken priorto and during treatment, is purified with the help of the RNeasy Kit(Qiagen, Hilden) and Bcl-2 mRNA levels are quantitated by real timeRT-PCR as described previously (Eder, M., et al., Leukemia (1999)13:1383-1389; Scherr, M., et al., BioTechniques (2001) 31:520-526).Analysis of Bcl-2 mRNA levels before and during treatment by real timePCR, provides the attending physician with a rapid and accurateassessment of treatment efficacy as well as the opportunity to modifythe treatment regimen in response to the patient's symptoms and diseaseprogression.

Example 6 dsRNA Expression Vectors

In another aspect of the invention, Bcl-2 specific dsRNA molecules thatinteract with Bcl-2 target RNA molecules and modulate Bcl-2 geneexpression activity are expressed from transcription units inserted intoDNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10;Skillem, A., et al., International PCT Publication No. WO 00/22113,Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S.Pat. No. 6,054,299). These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can beincorporated and inherited as a transgene integrated into the hostgenome. The transgene can also be constructed to permit it to beinherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl.Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. In apreferred embodiment, a dsRNA is expressed as an inverted repeat joinedby a linker polynucleotide sequence such that the dsRNA has a stem andloop structure.

The recombinant dsRNA expression vectors are preferably DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. NatI. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. NatI. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. NatI. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. NatI. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. NatI. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or U1 snRNA promoter) or preferably RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from a T7 promoter.The promoter can also direct transgene expression to the pancreas (see,e.g. the insulin regulatory sequence for pancreas (Bucchini et al.,1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Preferably, recombinant vectors capable of expressing dsRNA moleculesare delivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single target gene or multiple target genes over a period of a weekor more are also contemplated by the present invention. Successfulintroduction of the vectors of the invention into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofex vivo cells can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

The dsRNA 1 and 2 molecules can also be inserted into vectors and usedas gene therapy vectors for human patients. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al., Proc. Natl. Acad. Sci. USA91:3054-3057, 1994). The pharmaceutical preparation of the gene therapyvector can include the gene therapy vector in an acceptable diluent, orcan comprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

Example 7 Method of Determining an Effective Dose of a dsRNA

A therapeutically effective amount of a composition containing asequence that encodes Bcl-2 specific dsRNA, (i.e., an effective dosage),is an amount that inhibits expression of the polypeptide encoded by theBcl-2 target gene by at least 10 percent. Higher percentages ofinhibition, e.g., 15, 20, 30, 40, 50, 75, 85, 90 percent or higher maybe preferred in certain embodiments. Exemplary doses include milligramor microgram amounts of the molecule per kilogram of subject or sampleweight (e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). The compositions can be administered one time per week forbetween about 1 to 10 weeks, e.g., between 2 to 8 weeks, or betweenabout 3 to 7 weeks, or for about 4, 5, or 6 weeks. The skilled artisanwill appreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a composition can include a single treatment or a series oftreatments. In some cases transient expression of the dsRNA may bedesired. When an inducible promoter is included in the constructencoding an dsRNA, expression is assayed upon delivery to the subject ofan appropriate dose of the substance used to induce expression.

Appropriate doses of a composition depend upon the potency of themolecule (the sequence encoding the dsRNA) with respect to theexpression or activity to be modulated. One or more of these moleculescan be administered to an animal (e.g., a human) to modulate expressionor activity of one or more target polypeptides. A physician may, forexample, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular subject will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

The efficacy of treatment can be monitored either by measuring theamount of the Bcl-2 target gene mRNA (e.g. using real time PCR) or theamount of polypeptide encoded by the target gene mRNA (Western blotanalysis). In addition, the attending physician will monitor thesymptoms associated with pancreatic cancer afflicting the patient andcompare with those symptoms recorded prior to the initiation of dsRNAtreatment.

1-17. (canceled)
 18. A method for inhibiting the expression of the bcl-2gene in a cell, the method comprising: (a) introducing into the cell adouble-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises atleast two sequences that are complementary to each other and wherein asense strand comprises a first sequence and an antisense strandcomprises a second sequence comprising a region of complementarity whichis substantially complementary to at least a part of a mRNA encodingbcl-2, and wherein said region of complementarity is less than 30nucleotides in length and wherein said dsRNA, upon contact with a cellexpressing said bcl-2, inhibits expression of said bcl-2 gene by atleast 20%; and (b) maintaining the cell produced in step (a) for a timesufficient to obtain degradation of the mRNA transcript of the bcl-2gene, thereby inhibiting expression of the target gene in the cell. 19.A method of suppressing growth of a cancer cell, comprising contactingthe cell with a dsRNA, wherein the dsRNA comprises at least twosequences that are complementary to each other and wherein a sensestrand comprises a first sequence and an antisense strand comprises asecond sequence comprising a region of complementarity which issubstantially complementary to at least a part of a mRNA encoding bcl-2,and wherein said region of complementarity is less than 30 nucleotidesin length and wherein said dsRNA, upon contact with a cell expressingsaid bcl-2, inhibits expression of said bcl-2 gene by at least 20%. 20.A method of treating, preventing or managing cancer comprisingadministering to a patient in need of such treatment, prevention ormanagement a therapeutically or prophylactically effective amount ofdsRNA, wherein the dsRNA comprises at least two sequences that arecomplementary to each other and wherein a sense strand comprises a firstsequence and an antisense strand comprises a second sequence comprisinga region of complementarity which is substantially complementary to atleast a part of a mRNA encoding bcl-2, and wherein said region ofcomplementarity is less than 30 nucleotides in length and wherein saiddsRNA, upon contact with a cell expressing said bcl-2 inhibitsexpression of said bcl-2 gene by at least 20%. 21-22. (canceled)
 23. Themethod of claim 20, wherein said dsRNA is administered to treatpancreatic carcinoma.
 24. The method of claim 20, wherein said dsRNA isadministered by intraparenteral infusion.
 25. The method of claim 20,wherein said dsRNA is administered by intravenously.
 26. The method ofclaim 20, wherein said dsRNA is administered by injection.
 27. Themethod of claim 18, wherein said dsRNA comprises a sense strandconsisting of SEQ ID NO:29 and an antisense strand consisting of SEQ IDNO:30.
 28. The dsRNA of claim 27, wherein said dsRNA comprises at leastone modified nucleotide.
 29. The dsRNA of claim 28, wherein saidmodified nucleotide is chosen from the group of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group.
 30. The dsRNA of claim 28, wherein saidmodified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a2′-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.
 31. Themethod of claim 19, wherein said dsRNA comprises a sense strandconsisting of SEQ ID NO:29 and an antisense strand consisting of SEQ IDNO:30.
 32. The dsRNA of claim 31, wherein said dsRNA comprises at leastone modified nucleotide.
 33. The dsRNA of claim 32, wherein saidmodified nucleotide is chosen from the group of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group.
 34. The dsRNA of claim 32, wherein saidmodified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a2′-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.
 35. Themethod of claim 20, wherein said dsRNA comprises a sense strandconsisting of SEQ ID NO:29 and an antisense strand consisting of SEQ IDNO:30.
 36. The dsRNA of claim 35, wherein said dsRNA comprises at leastone modified nucleotide.
 37. The dsRNA of claim 36, wherein saidmodified nucleotide is chosen from the group of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group.
 38. The dsRNA of claim 36, wherein saidmodified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a2′-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.